Lipid metabolism is often elevated in cancer cells and plays an important role in their growth and malignancy. Acyl-CoA synthetase (ACS), which converts long-chain fatty acids to acyl-CoA, is overexpressed in various types of cancer. However, the role of ACS in cancer remains unknown. Here, we found that ACS enzyme activity is required for cancer cell survival. Namely, the ACS inhibitor Triacsin c induced massive apoptosis in glioma cells while this cell death was completely suppressed by overexpression of ACSL5, the Triacsin c-resistant ACS isozyme, but not by overexpression of a catalytically inactive ACSL5 mutant. ACS inhibition by Triacsin c markedly potentiated the Bax-induced intrinsic apoptotic pathway by promoting cytochrome c release and subsequent caspase activation. These effects were abrogated by ACSL5 overexpression. Correspondingly, ACS inhibition synergistically potentiated the glioma cell death induced by etoposide, a well-known activator of apoptosis. Furthermore, in a nude mouse xenograft model, Triacsin c at a non-toxic dose enhanced the antitumor efficacy of a low-dose chemotherapy with etoposide. These results indicate that ACS is an apoptosis suppressor and that ACS inhibition could be a rational strategy to amplify the antitumor effect of etoposide.