NEDD8 is an ubiquitin-like protein that is covalently attached to a lysine residue of a protein substrate through a process called neddylation, catalyzed by the enzyme cascade 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), the largest family of E3 ligases, whereas neddylation of non-cullin substrates alters their stability and activity, as well as subcellular localization. The neddylation pathway and/or many neddylation substrates are abnormally activated or over-expressed in various human diseases, such as metabolic disorders, liver dysfunction, neurodegenerative disorders, and cancers. Targeting neddylation becomes an attractive strategy for the treatment of these diseases. This review provides a general introduction on the neddylation cascade, its biochemical process and regulation, and the crystal structures of neddylation enzymes in complex with cullin substrates. It discusses how neddylation governs various key biological processes via the modification of cullins and non-cullin substrates. The review further reviews the literature data on dysregulated neddylation in several human diseases, particularly cancer, followed by an outline of current efforts in the discovery of small molecule inhibitors of neddylation as a promising therapeutic approach. Finally, few perspectives were proposed for extensive future investigations. The neddylation pathway is essential for the viability of most organisms and is involved in the development of several human diseases, including neurodegenerative disorders and cancers. The neddylation cascade consists of a single E1 (NAE), two E2s (UBE2F and UBE2M), and a few E3s, catalyzing a reaction to add the NEDD8 to its substrates. Deneddylation is a reverse reaction to remove NEDD8 from a neddylated substrate, catalyzed by an NEDD8 isopeptidase, such as COP9 signalosome complex (CSN). NEDD8 was first cloned in 1992 as a highly expressed gene in mouse neural precursor cells and characterized in 1993 to encode a small new peptide with 81 amino acids, sharing 59% identical and 80% homologous to ubiquitin. The neddylation pathway is crucial for the regulation of various biological processes and its dysregulation is associated with several human diseases. The neddylation pathway is also involved in the regulation of stress responses, including DNA damage response and nucleolar stress. The neddylation pathway plays a significant role in modulating DNA damage responses and ribosomal stress. The neddylation pathway is alsoNEDD8 is an ubiquitin-like protein that is covalently attached to a lysine residue of a protein substrate through a process called neddylation, catalyzed by the enzyme cascade 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), the largest family of E3 ligases, whereas neddylation of non-cullin substrates alters their stability and activity, as well as subcellular localization. The neddylation pathway and/or many neddylation substrates are abnormally activated or over-expressed in various human diseases, such as metabolic disorders, liver dysfunction, neurodegenerative disorders, and cancers. Targeting neddylation becomes an attractive strategy for the treatment of these diseases. This review provides a general introduction on the neddylation cascade, its biochemical process and regulation, and the crystal structures of neddylation enzymes in complex with cullin substrates. It discusses how neddylation governs various key biological processes via the modification of cullins and non-cullin substrates. The review further reviews the literature data on dysregulated neddylation in several human diseases, particularly cancer, followed by an outline of current efforts in the discovery of small molecule inhibitors of neddylation as a promising therapeutic approach. Finally, few perspectives were proposed for extensive future investigations. The neddylation pathway is essential for the viability of most organisms and is involved in the development of several human diseases, including neurodegenerative disorders and cancers. The neddylation cascade consists of a single E1 (NAE), two E2s (UBE2F and UBE2M), and a few E3s, catalyzing a reaction to add the NEDD8 to its substrates. Deneddylation is a reverse reaction to remove NEDD8 from a neddylated substrate, catalyzed by an NEDD8 isopeptidase, such as COP9 signalosome complex (CSN). NEDD8 was first cloned in 1992 as a highly expressed gene in mouse neural precursor cells and characterized in 1993 to encode a small new peptide with 81 amino acids, sharing 59% identical and 80% homologous to ubiquitin. The neddylation pathway is crucial for the regulation of various biological processes and its dysregulation is associated with several human diseases. The neddylation pathway is also involved in the regulation of stress responses, including DNA damage response and nucleolar stress. The neddylation pathway plays a significant role in modulating DNA damage responses and ribosomal stress. The neddylation pathway is also