Updated project metadata. O-GlcNAc is a nutritionally and metabolically relevant post-translational modification on thousands of nucleocytoplas-mic proteins. O-GlcNAcylation level dynamically responds to environmental cues in temporal and spatial dimensions, leading to discrete signaling transductions and physiological effects. Spatiotemporal regulation of O-GlcNAcylation level is essential for O-GlcNAc functional interrogation and manipulation of cell behaviors for desired outcomes, which is yet challenging owing to limited approaches. Here we achieved spatiotemporal O-GlcNAc reduction in live cells by designing a 4-hydroxytamoxifen (4-HT)-triggered O-GlcNAcase (OGA) activation strategy. After rational engineering and optimiza-tion, OGA variants bearing an intein located to different subcellular regions were generated and validated, whose deglyco-sidase activity can be turned on by 4-HT in a time- and dose-dependent manner. Finally, we demonstrated the dual func-tionality of 4-HT on breast cancer cells expressing the engineered OGA, which accelerated cell death with a lower dosage and thus mitigated the likelihood of acquiring drug resistance. Altogether, our strategy facilitates the precise regulation and functional understandings of O-GlcNAcylation in live cells.