NEDD8 (Neural precursor cell expressed developmentally downregulated protein 8) is an ubiquitin-like protein that undergoes neddylation, a process catalyzed by the enzyme cascade of NEDD8 activating enzyme (E1), NEDD8 conjugating enzyme (E2), and NEDD8 ligase (E3). Neddylation substrates are categorized into cullins and non-cullin proteins. Neddylation of cullins activates Cullin RING ligases (CRLs), while neddylation of non-cullin substrates alters their stability, activity, and subcellular localization. Abnormal activation or over-expression of the neddylation pathway and its substrates are associated with various human diseases, including metabolic disorders, liver dysfunction, neurodegenerative disorders, and cancers. This review provides an overview of the neddylation cascade, its biochemical process, and regulation, as well as the crystal structures of neddylation enzymes in complex with cullin substrates. It discusses how neddylation governs key biological processes and reviews the literature on dysregulated neddylation in human diseases, particularly cancer. The review also outlines current efforts in discovering small molecule inhibitors of neddylation as a promising therapeutic approach and proposes future perspectives for extensive investigations.NEDD8 (Neural precursor cell expressed developmentally downregulated protein 8) is an ubiquitin-like protein that undergoes neddylation, a process catalyzed by the enzyme cascade of NEDD8 activating enzyme (E1), NEDD8 conjugating enzyme (E2), and NEDD8 ligase (E3). Neddylation substrates are categorized into cullins and non-cullin proteins. Neddylation of cullins activates Cullin RING ligases (CRLs), while neddylation of non-cullin substrates alters their stability, activity, and subcellular localization. Abnormal activation or over-expression of the neddylation pathway and its substrates are associated with various human diseases, including metabolic disorders, liver dysfunction, neurodegenerative disorders, and cancers. This review provides an overview of the neddylation cascade, its biochemical process, and regulation, as well as the crystal structures of neddylation enzymes in complex with cullin substrates. It discusses how neddylation governs key biological processes and reviews the literature on dysregulated neddylation in human diseases, particularly cancer. The review also outlines current efforts in discovering small molecule inhibitors of neddylation as a promising therapeutic approach and proposes future perspectives for extensive investigations.