objective:to develop a 5-fluorouracil(5-fu)-resistant human pancreatic cancer cell line patu8988/5-fu and investigate its biological characteristics and the preliminary mechanisms of chemoresistance in pancreatic cancer. methods:patu8988 was exposured to increased 5-fu progressively for 10 months to obtain stable 5-fu-resistant patu8988/5-fu in vitro. the morphological features and growth characters of patu8988/5-fu were observed. mtt assay was used to observe its sensitivity of drug resistance and cross resistance. the growth curve was observed and the doubling time was calculated. flow cytometry assay was used to evaluate the cell cycle distribution,apoptosis rate and accumulation of rh123 in two cell lines. the expressions of hif-1α and mdr1 mrna were detected by rt-pcr. results:the acquired multidrug resistance(mdr) cell line of patu8988/5-fu was established successfully. compared with the parental cell,patu8988/5-fu showed great changes in biological characteristics. the drug resistance indexes of cell line patu8988/5-fu to 5-fu, adriamycin (adm) and gemcitabine were 36.98,2.76 and 2.42,respectively. compared with the parental cell,patu8988/5-fu cell exhibited a lower growth rate,prolonged population doubling time,and cell distribution decreased in s phase and increased in g2/m phase. the expressions of hif-1α and mdr1 mrna were significantly higher,while the accumulation of rh123 in cytoplasm and the apoptotic ratio after treated with different concentration of 5-fu were significantly lower in patu8988/5-fu cell than in the parental cell. conclusion:the acquired mdr cell line of patu8988/5-fu was established successfully after exposure to increased 5-fu. hif1α maybe contribute to acquirement of drug resistance by inhibiting apoptosis and upregulating the expression of the main drug resistant related genes mdr1.