This study focuses on exploring the role of Six2 in the progression of hepatocellular carcinoma (HCC) and its resistance to the chemotherapy drug 5-fluorouracil (5-FU). Using Hep3B and Huh7 cell lines, we analyzed how Six2 affects various cellular functions, including viability, proliferation, apoptosis, and invasion. Our research also delved into Six2's regulatory impact on DNMT1 levels, E-cadherin expression, and the methylation of the E-cadherin promoter, all of which are crucial for 5-FU resistance in HCC cells. Additionally, we examined the effects of Six2 knockdown on the PI3K/AKT/mTOR signaling pathway. Our findings indicate that overexpression of Six2 enhances cell viability and proliferation, encourages invasive behavior, increases methylation at the E-cadherin promoter, and reduces apoptosis. These changes correspond with increased levels of DNMT1 and decreased levels of E-cadherin, culminating in heightened resistance to 5-FU. Conversely, knocking down Six2 increases the sensitivity of HCC cells to 5-FU and reduces activation of the PI3K/AKT/mTOR pathway. These results suggest that Six2 plays a significant role in promoting HCC proliferation, invasion, and chemotherapy resistance, particularly through mechanisms involving DNMT1 and the PI3K/AKT/mTOR pathway, highlighting its potential as a target for HCC treatment.