Gemcitabine (Gem) is currently the first-line chemotherapeutic drug in management of pancreatic cancer, however the therapeutic efficacy of Gem is limited due to its short half-life and poor cell membrane permeability. Here we designed mesoporous silica vesicles (MSVs) with large pore sizes as a novel drug delivery system. The MSVs were synthesized using cetyltrimethyl ammonium bromide (CTAB) as a structure-directing agent, tetraethoxysilane (TEOS) as silica source in n-hexane/water biliquid system. By virtue of the large pore size and large pore volume of the MSVs, Gem was loaded into the mesoporous of MSVs via “nanocasting” method. In vitro drug release experiments of gemcitabine-loaded MSVs showed an accelerating release of gemcitabine in acidic condition. These fluorescently labeled MSVs could be effectively internalized by both a human (BxPC-3) and a mouse pancreatic cancer cell lines (Pan02). Additionally, some MSVs could even reach the nuclei of the pancreatic cancer cells. Cell viability assays demonstrated that gemcitabine-loaded MSVs exhibited enhanced anticancer activity in inhibiting the proliferation of BxPC-3 and Pan02 cells compared with free Gem, while the MSVs alone showed no significant cytotoxicity. Our results indicate that our synthesized MSVs might represent a promising novel drug delivery platform for the treatment of pancreatic cancer.