O-GlcNAc transferase (OGT) plays an important role in regulating numerous cellular processes through reversible post-translational modification of nuclear and cytoplasmic proteins. However, the function of O-GlcNAcylation is still not well understood. Cell permeable OGT inhibitors are needed to manipulate O-GlcNAcylation levels and clarify the regulatory mechanism of this modification. Here, we report a specific natural-product OGT inhibitor (L01), which was identified from a structure-based virtual screening analysis. L01 inhibited O-GlcNAcylation both in vitro and in cells without significantly altering cell surface glycans. Molecular dynamics and site-directed mutagenesis indicated a new binding mechanism in which L01 could interact with Asn557 near the UDP binding pocket of OGT. This residue may contribute to the specificity of L01. Furthermore, as a specific OGT inhibitor, L01 produced low toxicity in cellular and zebrafish models. The identification of L01 validates structure-based virtual screening approaches for the discovery of OGT inhibitors. L01 can also serve as a chemical tool to further characterize O-GlcNAcylation functions or a new molecular core for structure-activity relationship studies to optimize the biochemical potencies.