BRD4 undergoes liquid-liquid phase separation (LLPS) at super-enhancers (SEs) and confers prominent gene control. However, the regulatory mechanism and transcriptional consequences of BRD4 condensation remain elusive. Here we identified that the C-terminal intrinsic disordered region of BRD4 serves as the primary interacting region with O-GlcNAc transferase (OGT) for O-GlcNAc modifications at 12 possible sites. O-GlcNAcylation attenuates the LLPS propensity of BRD4 with reduced puncta size and increased fluidity. These in turn result in significantly declined occupancy and transcription at SEs. We further demonstrated that loss of O-GlcNAcylation on BRD4 led to elevated gene expression relating to cell cycle and DNA damage, allowing cells less sensitive to DNA damage and grow faster. Therefore, our results demonstrate an O-GlcNAc–mediated regulatory mechanism of BRD4 LLPS for gene control at SEs, showcasing an example of how post-translational modifications regulate transcriptional activities at SEs through a phase separation model.