Transglutaminases (Tgases) are a diverse group of enzymes that catalyze post-translational modifications of proteins by forming isopeptide bonds, either through protein cross-linking via ε-(γ-glutamyl)lysine bonds or by incorporating primary amines at selected peptide-bound glutamine residues. These cross-linked products are highly resistant to mechanical stress and proteolysis and are found in various tissues and processes, including skin, hair, blood clotting, and wound healing. However, deregulation of Tgase activity can lead to several human diseases, such as chronic neurodegeneration, neoplastic diseases, autoimmune diseases, and tissue fibrosis. The review focuses on the structural and regulatory features of mammalian Tgases, particularly the ubiquitous type 2 tissue Tgase (tTgase), which is a multifunctional protein with both protein-cross-linking and GTP-hydrolyzing activities. The enzyme's activation by Ca2+ and its regulation by GTP are discussed, along with its physiological roles and substrates. The commercial applications of Tgases, particularly in biotechnology, are also highlighted. The review aims to enhance understanding of the pathogenesis of diseases associated with Tgases and to explore their potential in biotechnological applications.Transglutaminases (Tgases) are a diverse group of enzymes that catalyze post-translational modifications of proteins by forming isopeptide bonds, either through protein cross-linking via ε-(γ-glutamyl)lysine bonds or by incorporating primary amines at selected peptide-bound glutamine residues. These cross-linked products are highly resistant to mechanical stress and proteolysis and are found in various tissues and processes, including skin, hair, blood clotting, and wound healing. However, deregulation of Tgase activity can lead to several human diseases, such as chronic neurodegeneration, neoplastic diseases, autoimmune diseases, and tissue fibrosis. The review focuses on the structural and regulatory features of mammalian Tgases, particularly the ubiquitous type 2 tissue Tgase (tTgase), which is a multifunctional protein with both protein-cross-linking and GTP-hydrolyzing activities. The enzyme's activation by Ca2+ and its regulation by GTP are discussed, along with its physiological roles and substrates. The commercial applications of Tgases, particularly in biotechnology, are also highlighted. The review aims to enhance understanding of the pathogenesis of diseases associated with Tgases and to explore their potential in biotechnological applications.