Objective：This study aims to investigate the role of aquaporin 3（AQP3） in the chemoresistance of colorectal carcinoma mediated by hypoxia. Methods：Real-time quantitative PCR and Western blot were performed to detect the expression of AQP3 under hypoxic condition in colorectal carcinoma HCT116 cells and HT29 cells. CCK-8 was used to detect the influence of hypoxia on the sensitivity of HCT116 cells to 5-fluorouracil（5-Fu）under hypoxic condition. CCK-8 and colony formation assay were also used to detect the effect of AQP3 on the sensitivity of HCT116 cells to 5-Fu under hypoxic condition. Results：Under hypoxic condition，the expression levels of AQP3 mRNA and protein were significantly up-regulated in both HCT116 and HT29 cells. After hypoxia inducible factor（HIF-1α）was inhibited，and the expression levels of AQP3 mRNA and protein in HCT116 cells under hypoxic condition were not significantly increased. Under hypoxic condition，the sensitivity of HCT116 cells to 5-Fu was lower than that in normal oxygen condition，which was reversed by inhibiting AQP3. Colony formation assay also demonstrated that the effect of 5-Fu on the proliferation ability of HCT116 cells was smaller under hypoxic condition，which was reversed by inhibiting AQP3. Conclusion：Under hypoxic condition，colorectal carcinoma up-regulated AQP3 expression by HIF-1α，which lead to 5-Fu chemoresistance.