Depression presents challenges in understanding its biological basis and developing effective treatments. Posttranslational modifications provide critical insights into molecular mechanisms of disorders, yet their role in depression remains underexplored. Glycosylation has been particularly difficult to study due to its heterogeneous nature and analytical challenges, despite its potential to uncover previously unknown mechanisms. In this study, we demonstrate how chronic stress alters region-specific O-glycosylation across multiple brain regions, with a particular focus on the prefrontal cortex (PFC). Our findings reveal significant changes in sialylated O-glycosylation patterns mediated by St3gal1, a sialyltransferase essential for O-glycosylation. Notably, knockdown of St3gal1 in nonstressed mice induced depressive-like behaviors, whereas its overexpression in stressed mice alleviated depressive symptoms, underscoring its regulatory role in stress resilience. In addition, we identify potential glycoprotein targets and downstream regulators of St3gal1, including neurexin 2 (NRXN2) in the medial PFC (mPFC), offering insights into molecular pathways linking O-glycosylation to depressive-like behaviors.