The aim of the study was to observe the effect of small interference RNA (siRNA) targeting hypoxia-inducible factor 1α (HIF-1α) combined L-ascorbate on proliferation, migration, and apoptosis of hypoxic MiaPaCa2 human pancreatic cancer cells. A cassette encoding siRNA targeting HIF-1α mediated by recombinant adeno-associated virus (rAAV) was constructed, giving rAAV-siHIF. rAAV-siHIF and L-ascorbate, which were used alone or in combination, were delivered to exponentially growing MiaPaCa2 cells under hypoxic conditions. Then, we observed the expression of HIF-1α mRNA and protein, the proliferation, apoptosis, and migration of MiaPaCa2 cells by real-time PCR, Western blot, MTT, TUNEL, and Transwell assay, respectively. Under hypoxic conditions, rAAV-siHIF inhibited the expression of HIF-1α mRNA in MiaPaCa2 cells but L-ascorbate did not. However, rAAV-siHIF and L-ascorbate both inhibited the expression of HIF-1α protein and the proliferation and migration of MiaPaCa2 cells and induced MiaPaCa2 cell apoptosis. The effect in the combined group was more efficient than that seen when rAAV-siHIF or L-ascorbate was used separately. rAAV-siHIF and L-ascorbate both affect biological behavior of hypoxic MiaPaCa2 cells through modulating HIF-1α protein expression and rAAV-siHIF and L-ascorbate have synergy under hypoxic conditions.