Small-molecule Bcl-2/Bcl-xL inhibitor Navitoclax represents a promising cancer therapeutic since preclinical and clinical studies with Navitoclax have demonstrated strong anticancer activity in several types of cancers. However, because Navitoclax has a low binding affinity to Mcl-1, anticancer activity by Navitoclax is often attenuated by the elevated expression of Mcl-1 in hepatocellular carcinoma (HCC) and other cancers, posing a serious problem for its potential clinical utilities. Therefore, approaches that suppress the expression of Mcl-1 are urgently needed to overcome Navitoclax-resistance in these cancers. Here, we reported that aspirin markedly suppressed Mcl-1 expression, and significantly enhanced Navitoclax-mediated cell viability inhibition and apoptosis induction in HCC cells. We further showed that aspirin robustly enhanced Navitoclax-triggered cytosolic cytochrome c release, activation of initiator caspase-9 and effector caspase-3, and cleavage of PARP. Importantly, the cell death induction by the combination could be rescued by a cell-permeable caspase-9 inhibitor Z-LEHD-FMK, indicative of an indispensable role of mitochondrial apoptosis pathway during the combination effect. Taken together, our study suggests that aspirin can be used to enhance Navitoclax-mediated anticancer activity via suppression of Mcl-1. Since aspirin is one of the most commonly used medicines, our findings therefore have translational impacts on Navitoclax-based therapy for HCC. (C) 2013 Elsevier Inc. All rights reserved.