Protein glycosylation is a widespread post-translational modification found in all living organisms. Despite their complexity in animals, glycan structures play crucial biological and physiological roles, from protein folding and quality control to biological recognition events. Advances in analytical methods and biochemical approaches have improved understanding of the biological roles of these complex structures in vertebrates. Glycans associated with cell surface and intracellular proteins and lipids contribute to numerous biological functions in animals. Oligosaccharide structures at the cell surface influence interactions with the extracellular environment by providing ligands for cell adhesion, macromolecule interactions, and pathogen invasion. Glycans associated with cell surface receptors and proteins also directly modulate protein function and signaling, as well as altering the dynamics of glycoprotein endocytosis and cell surface half-life through binding to multivalent lectins. Glycan structures on newly synthesized glycoproteins are crucial for protein secretion, as they influence protein folding, provide ligands for lectin chaperones, contribute to quality control surveillance in the endoplasmic reticulum (ER), and mediate transit and selective protein targeting throughout the secretory pathway. Glycan structures also contribute to the regulation of cytosolic and nuclear functions, immune surveillance, inflammatory reactions, autoimmunity, hormone action, and tumor metastasis. Protein glycosylation is widely used by cell biologists to monitor protein transit through the secretory pathway. Glycans detected by specific antibodies and ectopically expressed fluorescently tagged glycosylation enzymes are used as markers for intracellular compartments. Glycan structures also distinguish cell types in developing and mature animal tissues. Despite this limited use of mammalian glycan structures or glycosylation machinery as tools for cell biology studies, few cell biologists have truly embraced the diversity and complexity of glycan modifications for their contributions and roles in biological systems. This results from an intimidating collection of challenges for studying the functions of glycan structures at the molecular, cellular, and organismal level. It has been estimated that approximately 700 proteins are required to generate the full diversity of mammalian glycans (estimated to be ≥7,000 structures), which are assembled from only ten monosaccharides. Among these proteins, ~200 are glycosyltransferases, which are enzymes that extend acceptor glycan structures using nucleotide or lipid-linked sugars as activated donor substrates. Competition between glycosyltransferases that possess overlapping glycan acceptor preferences but different donor specificities can strongly influence the relative abundance of glycan structural features in the total glycome of a cell or tissue. Glycans can be attached to polypeptide structures through amide linkages to Asn side chains (N-glycosylation), through glycosidic linkages (O-glycosylation) to side chains of Ser/Thr, hydroxyllysine (collagen) orProtein glycosylation is a widespread post-translational modification found in all living organisms. Despite their complexity in animals, glycan structures play crucial biological and physiological roles, from protein folding and quality control to biological recognition events. Advances in analytical methods and biochemical approaches have improved understanding of the biological roles of these complex structures in vertebrates. Glycans associated with cell surface and intracellular proteins and lipids contribute to numerous biological functions in animals. Oligosaccharide structures at the cell surface influence interactions with the extracellular environment by providing ligands for cell adhesion, macromolecule interactions, and pathogen invasion. Glycans associated with cell surface receptors and proteins also directly modulate protein function and signaling, as well as altering the dynamics of glycoprotein endocytosis and cell surface half-life through binding to multivalent lectins. Glycan structures on newly synthesized glycoproteins are crucial for protein secretion, as they influence protein folding, provide ligands for lectin chaperones, contribute to quality control surveillance in the endoplasmic reticulum (ER), and mediate transit and selective protein targeting throughout the secretory pathway. Glycan structures also contribute to the regulation of cytosolic and nuclear functions, immune surveillance, inflammatory reactions, autoimmunity, hormone action, and tumor metastasis. Protein glycosylation is widely used by cell biologists to monitor protein transit through the secretory pathway. Glycans detected by specific antibodies and ectopically expressed fluorescently tagged glycosylation enzymes are used as markers for intracellular compartments. Glycan structures also distinguish cell types in developing and mature animal tissues. Despite this limited use of mammalian glycan structures or glycosylation machinery as tools for cell biology studies, few cell biologists have truly embraced the diversity and complexity of glycan modifications for their contributions and roles in biological systems. This results from an intimidating collection of challenges for studying the functions of glycan structures at the molecular, cellular, and organismal level. It has been estimated that approximately 700 proteins are required to generate the full diversity of mammalian glycans (estimated to be ≥7,000 structures), which are assembled from only ten monosaccharides. Among these proteins, ~200 are glycosyltransferases, which are enzymes that extend acceptor glycan structures using nucleotide or lipid-linked sugars as activated donor substrates. Competition between glycosyltransferases that possess overlapping glycan acceptor preferences but different donor specificities can strongly influence the relative abundance of glycan structural features in the total glycome of a cell or tissue. Glycans can be attached to polypeptide structures through amide linkages to Asn side chains (N-glycosylation), through glycosidic linkages (O-glycosylation) to side chains of Ser/Thr, hydroxyllysine (collagen) or