Gallbladder cancer (GBC) is the most common malignant tumor of the biliary tract and is often prone to early distant metastasis. However, the mechanisms underlying GBC's invasive metastasis remain unclear. This study identified that F-box only protein 33 (FBXO33) expression is significantly elevated in GBC and is negatively associated with patient prognosis. In vivo and in vitro experiments demonstrated that knockdown of FBXO33 inhibits epithelial-mesenchymal transition (EMT) progression in GBC, while overexpression of FBXO33 promotes EMT progression. Mechanistically, FBXO33 regulates EMT progression by modulating the polyubiquitination of p53 at K291 and K292. Moreover, the upregulation of FBXO33 in GBC is driven by transcriptional regulation mediated by Yin Yang-1 (YY1). The lactylation modification of YY1 at K183 was found to be essential for the transcriptional activation of FBXO33. These findings underscore the role of the lactylation-driven FBXO33-p53 axis in promoting the invasive metastasis of GBC.