Ethanol is a common risk factor for liver injury. Cucurbitacin B (CuB) is a natural product with potent cytotoxic activities mediated by inducing apoptosis. This study investigated the effect of ethanol on CuB-induced cytotoxicity in LO2 hepatocytes. Low concentration of ethanol alone showed no significant cytotoxic effect on LO2 cells. CuB dose-dependently decreased cell viability. However, ethanol co-treatment significantly enhanced CuB-induced cytotoxicity. CuB-induced mitochondria membrane potential (ΔΨ) depolarization was further decreased by ethanol. Furthermore, CuB-induced apoptosis was augmented by ethanol, as evidenced by DNA fragmentation, Annexin V staining, and apoptotic protein expression. Ethanol inhibited CuB-induced autophagy, as determined by MDC staining, autophagic protein expression, and transmission electron microscopy. Therefore, the present data suggested that ethanol enhanced CuB cytotoxicity in LO2 hepatocytes, which was mediated by inhibiting autophagy and augmenting apoptosis.