Chemotherapy continues to be a mainstay of cancer treatment, although drug resistance is a majorobstacle. Lipid metabolism plays a critical role in cancer pathology, with elevated ether lipid levels. Recently,alkylglyceronephosphate synthase (AGPS), an enzyme that catalyzes the critical step in ether lipid synthesis,was shown to be up-regulated in multiple types of cancer cells and primary tumors. Here, we demonstrated thatsilencing of AGPS in chemotherapy resistance glioma U87MG/DDP and hepatic carcinoma HepG2/ADM celllines resulted in reduced cell proliferation, increased drug sensitivity, cell cycle arrest and cell apoptosis throughreducing the intracellular concentration of lysophosphatidic acid (LPA), lysophosphatidic acid-ether (LPAe)and prostaglandin E2 (PGE2), resulting in reduction of LPA receptor and EP receptors mediated PI3K/AKTsignaling pathways and the expression of several multi-drug resistance genes, like MDR1, MRP1 and ABCG2.β-catenin, caspase-3/8, Bcl-2 and survivin were also found to be involved. In summary, our studies indicate thatAGPS plays a role in cancer chemotherapy resistance by mediating signaling lipid metabolism in cancer cells.