Chemotherapeutic drug resistance of tumor cells under hypoxic conditions is caused by the inhibition of apoptosis by autophagy and drug efflux via adenosine triphosphate (ATP)-dependent transporter activation, among other factors. Here, we demonstrate that disrupting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression can reduce the autophagy and ATP levels in tumor cells. To test whether GAPDH knockdown is sufficient to overcome drug resistance, a nanocarrier (asymmetry-membrane liposome) was designed to encapsulate GAPDH-siRNA with a low dose of paclitaxel (PTX). Liposomes were prepared using novel cryogenic inner-outer dual reverse phase emulsion liposome manufacturing technology to obtain a high loading of siRNA. The results of dynamic light scattering (DLS) indicated that the liposomes had an average hydrodynamic diameter of 250.5 nm and polydispersity index (PDI) of 0.210, which was confirmed by (Transmission Electron Microscope) TEM images. In in vitro tests, the siRNA liposomes presented a high specificity in the suppression of GAPDH expression and significant synergy in cytotoxicity with co-delivery of PTX against tumor cells (HeLa and MCF-7) under hypoxic conditions. Moreover, in vivo studies (a HeLa tumor xenograft model using female BALB/c nude mice) demonstrate that the liposomes could not only increase the concentration of drugs in tumors over time but also successfully boosted the chemotherapeutic efficacy of PTX (synergistic therapy with GAPDH-siRNA). Tumor cells appeared to lose their resistance against PTX therapy, becoming more sensitive to PTX when GAPDH-siRNA was simultaneously administered in long-circulating liposomes. Consequently, the novel delivery of GAPDH-siRNA using nanotargeted liposomes provides a useful and potential tool to overcome multidrug resistant (MDR) tumors and presents a bright prospect compared with the traditional chemotherapeutic strategies in clinic cancer therapy.