Mammalian cell cycle is a central process for tissue growth and maintenance. Protein O-linked β-N-acetylglucosamine (O-GlcNAc) modification has been found to occur on several important cell cycle regulators. However, the O-GlcNAcylated proteome has not been extensively profiled during cell cycle progression. Herein, we report a quantitative profiling of protein O-GlcNAcylation sites in cell proliferation, by using an O-GlcNAc chemoproteomic strategy. In HeLa cells, a total of 902, 439, and 872 high-confidence O-GlcNAcylation sites distributed on 414, 265, and 425 proteins are identified in the interphase, early mitosis, and mitotic exit stages, respectively. The identified O-GlcNAcylation events occur on a variety of important regulators, which are involved in the processes of cell division, DNA repair, and cell death. Furthermore, we show that O-GlcNAcylation is dynamically regulated in a cell cycle stage-dependent manner. Our results provide a valuable resource for investigating the functional roles of O-GlcNAc in the mammalian cell cycle.