O-GlcNAcylation, a recently discovered post-translational modification of proteins, plays a crucial role in regulating protein structure and function, and is closely associated with multiple diseases. Research has shown that O-GlcNAcylation is abnormally upregulated in most cancers, promoting disease progression. To elucidate the roles of O-GlcNAcylation in cancer, this review summarizes various cancer-associated biological events regulated by O-GlcNAcylation and the corresponding signaling pathways. This work may provide insights for future studies on the function or underlying mechanisms of O-GlcNAcylation in cancer.