Hypoxia is a hallmark of pancreatic cancer (PC) and is associated with gemcitabine resistance. However, the mechanisms underlying hypoxia-induced gemcitabine resistance in PC remain greatly unknown. Our previous work showed that miR-301a, a hypoxia-sensitive miRNA, is involved in PC metastasis under hypoxia via regulation of its target gene P63. Here, we showed that miR-301a was upregulated in a NF-κB independent manner and promoted gemcitabine resistance under hypoxic conditions in vitro. In addition, TAp63, a member of the P63 family, reversed hypoxia-induced gemcitabine resistance by promoting degradation of HIF-1α. Furthermore, we proved that TAp63 was a functional downstream target of miR-301a and mediated the biological properties of miR-301a in PC. Taken together, these findings indicate that miR-301a exerts as a critical regulator involved in hypoxia-induced gemcitabine resistance in PC and may have potentials to be a therapeutic target for PC patients.