Advanced colorectal cancer (CRC) is one of the deadliest cancers, and the 5-year survival rate of patients with metastasis is extremely low. The epithelial-mesenchymal transition (EMT) is considered essential for metastatic CRC, but the fundamental molecular basis underlying this effect remains unknown. Here, we identified that O-GlcNAcylation, a unique posttranslational modification (PTM) involved in cancer metabolic reprogramming, increased the metastatic capability of CRC. The levels of O-GlcNAcylation were increased in the metastatic CRC tissues and cell lines, which likely promoted the EMT by enhancing EZH2 protein stability and function. The CRC patients with higher levels of O-GlcNAcylation exhibited greater lymph node metastasis potential and lower overall survival. Bioinformatic analysis and luciferase reporter assays revealed that both O-GlcNAcylation transferase (OGT) and EZH2 are posttranscriptionally inhibited by microRNA-101. In addition, O-GlcNAcylation and H3K27me3 modification in the miR-101 promoter region further inhibited the transcription of miR-101, resulting in the upregulation of OGT and EZH2 in metastatic CRC, thus forming a vicious cycle. In this study, we demonstrated that O-GlcNAcylation, which is negatively regulated by microRNA-101, likely promotes CRC metastasis by enhancing EZH2 protein stability and function. Reducing O-GlcNAcylation may be a potential therapeutic strategy for metastatic CRC.