In order to construct plasmid of hypoxia-inducible factor-lalpha (HIF-1α), and transfect into human lung cancer cells A549, the change in sensitivity of lung cancer cells A549 to chemotherapy was observed. HIF-1α mRNA structure region was amplified by RT-PCR and inserted into plasmid pcDNA3. The expression plasmid pcDNA3/HIF-1α was transfected into A549 with LipofectAMINETM2000. The expression of HIF-1α protein was detected by West blot. After A549 cells were transfected with HIF-1α prior to addition of 5-Fu, the growth activity was measured by growth curve, apoptosis was detected by flow cytometry at 48 h, and the levels of caspase3 and MDR-1 were determined by West blot. The results showed that the constructed expression plasmid was analyzed with restriction enzymes and gel electrophoresis. Two DNA lanes at 2.55 kb and 5.4 kb respectively were found, which were consistent with that expected. The growth rate in 5-Fu group was significantly inhibited, and the apoptosis index and caspase3 activity were increased significantly as compared with control group. After HIF-1 α being transfected into A549, the activity of MDR-1 was increased and the effect of 5-Fu was weakened. In conclusion, HIF-1α can promote chemoresistance by increasing the activation of MDR1 and suppressing apoptosis during lung cancer cells A549 induced with 5-Fu.