The nutrient sensor O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification found on thousands of nucleocytoplasmic proteins. O-GlcNAc levels in cells dynamically respond to environmental cues in a temporal and spatial manner, leading to altered signal transduction and functional effects. The spatiotemporal regulation of O-GlcNAc levels would accelerate functional interrogation of O-GlcNAc and manipulation of cell behaviors for desired outcomes. Here, we report a strategy for spatiotemporal reduction of O-GlcNAc in live cells by designing an O-GlcNAcase (OGA) fused to an intein triggered by 4-hydroxytamoxifen (4-HT). After rational protein engineering and optimization, we identified an OGA-intein variant whose deglycosidase activity can be triggered in the desired subcellular compartments by 4-HT in a time- and dose-dependent manner. Finally, we demonstrated that 4-HT activation of the OGA-intein fusion can likewise potentiate inhibitory effects in breast cancer cells by virtue of the reduction of O-GlcNAc. The spatiotemporal control of O-GlcNAc through the chemically activatable OGA-intein fusion will facilitate the manipulation and functional understanding of O-GlcNAc in live cells.