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Activation of signal transducer and activator of transcription 3 (STAT3) is associated with hypoxia-induced epithelial-mesenchymal transition (EMT). Activation of STAT3 requires its phosphorylated form, and STAT3 can also be post-translationally modified by O-GlcNAcylation. Dynamic regulation of STAT3 O-GlcNAcylation in relation to STAT3 phosphorylation remains poorly understood. We observed, based on chemical enzyme labeling and click chemistry methods in combination with mass spectrometric analysis, that O-GlcNAcylation of STAT3 is significantly reduced under hypoxia. Results of functional experiments indicated that O-GlcNAcylation maintains stability of STAT3 and prevents its degradation via ubiquitination during hypoxia-induced EMT. O-GlcNAcylation of STAT3 facilitated its phosphorylation. Following STAT3 phosphorylation, existing STAT3 O-GlcNAcylation was antagonistically released. Our experimental findings, in combination with structure modeling, indicate that O-GlcNAcylation of STAT3 at residue T717 is essential for its phosphorylation at Y705. In contrast, mutation of STAT3 at phosphorylation site Y705 had no effect on its O-GlcNAcylation. O-GlcNAcylation and phosphorylation of STAT3 evidently occur in a strict sequential order under hypoxia-induced EMT. Dynamic regulation of STAT3 function clearly involves crosstalk between O-GlcNAcylation and phosphorylation. O-GlcNAcylation of STAT3 at T717 facilitates EMT process by promoting STAT3 phosphorylation, and provides a potential therapeutic target that may be useful in anticancer drug design., (Copyright © 2022 Elsevier Inc. All rights reserved.)