Hypoxia-inducible factor 1α (HIF1α) has emerged as a promising new target for pancreatic cancer treatment over the past decade. High expression of HIF-1α increases the drug resistance of the current first line chemotherapeutic drug, gemcitabine (Gem). Here we employed biocompatible lipid-polymer hybrid nanoparticles to co-deliver HIF1α siRNA (si-HIF1α) and Gem for pancreatic cancer treatment in subcutaneous and orthotopic tumor models. The cationic ε-polylysine co-polymer (ENPs) can effectively absorb negatively charged si-HIF1α on the surface and encapsulate Gem to the hydrophilic core. Further coating of ENPs with PEGylated lipid bilayer resulted formation of LENPs, with reversed surface charge. The lipid bilayer of LENPs prevented nanoparticle aggregation and si-HIF1α degradation in serum, as well as Gem leakage. Those characteristics endow LENPs encapsulating drug prolonged lifetime in bloodstream and improved drug release via the enhanced tumor vasculature effect in tumor tissues. LENPs can co-deliver Gem and si-HIF1α (LENP-Gem-si-HIF1α) into tumor cells and effectively suppress the HIF1α expression both in vitro and in vivo. LENP-Gem-siHIF1α exhibited significant synergistic antitumor effects. Furthermore, LENP-Gem-si-HIF1α showed excellent capability to inhibit tumor metastasis in orthotopic tumor model.