Updated publication reference for PubMed record(s): 31527085. O-GlcNAcylation occurs on thousands of proteins involved in various cellular events. O-GlcNAc transferase (OGT), one of the enzymes responsible for protein O-GlcNAcylation, is known to be auto-O-GlcNAcylated at multiple sites. However, the role of O-GlcNAcylation on OGT is still unknown. Here, we report the role of O-GlcNAcylation on short form OGT (sOGT). By LC-ETD-MS analysis and western blot, we show that sOGT was mainly O-GlcNAcylated in the tetratricopeptide repeat (TPR) motifs with T12 and S56 being two “key” sites. T12 was a predominant O-GlcNAcylation site and the absence of S56 O-GlcNAcylation enhanced sOGT O-GlcNAcylation level. We found that O-GlcNAcylation of sOGT did not affect the enzyme activity but increased its binding to substrate proteins. S56A bound to and hence glycosylated more proteins, while T12A associated with fewer substrates. Proteomic studies combined with cell proliferation/cell cycle analyses indicated that S56A substantially enhanced cell proliferation, while T12A reduced it, and T12A led to a G2/M cell cycle arrest, due to O-GlcNAcylation of diverse proteins. These findings demonstrate that the O-GlcNAc pattern on sOGT modulates its function in cells by targeting different proteins.