Objective:To investigate the effects of mitochondrial damage and drug resistance of 5-FU-resistant human pancreatic cancer cell line Patu8988/5-FU cultured by cobalt chloride(CoCl2). Methods:Patu8988/5-FU were cultured in medium containing different concentrations of CoCl2(0,6.25 μmol/L,12.50 μmol/L,25.00 μmol/L,50.00 μmol/L,100.00 μmol/L,200.00 μmol/L,400.00 μmol/L). MTT assay was used to detect the cell proliferation activity and drug resistance of Patu8988/5-FU. JC-1 fluorescence staining with flow cytometry was used to analyze the mitochondria membrane potential. The mRNA expressions of HIF-1α and MDR1 were detected by RT-PCR technique. P-gp pump function of Patu8988/5-FU cells’ membrane was tested by Rh123. Results:With CoCl2 concentration rising or 100 μmol/L CoCl2 treatment time extension,the proliferation activity of Patu8988/5-FU cells was degressive,the depolarization of mitochondrial membrane potential was increased. Drug resistance index of Patu8988/5-FU to 5-FU in normoxia and hypoxia reaches to 28.11±3.19 and 52.08 ± 3.53,respectively(P < 0.01). The mRNA expressions of HIF-1α and MDR1 in Patu8988/5-FU cells is higher in hypoxia than in normoxia (P < 0.01). The accumulation amount of Rh123 in cytoplasm of Patu8988/5-FU cells was significantly lower in hypoxia than in normoxia(P < 0.01). Conclusion:CoCl2 induced chemistry hypoxia of Patu8988/5-FU cells in a time-and-dose dependent manner. CoCl2 mimicing hypoxia dramatically increased resistance of Patu8988/5-FU cells to 5-FU,and maybe through upregulating the expression of HIF-1α and MDR1.