Valproic acid (VPA), which is clinically use of as an anticonvulsant and mood-stabilizing drug, has been widely studied in the treatment of various cancers. Liriodenine (Li) is an aporphine compound extracted from the leaves of Michelia compressa. In this study, we evaluated a potential synergistic effect of Li and VPA on apoptosis in human colon cancer cell lines. Compared with the use of Li alone or VPA alone, Li/VPA synergistically induced apoptosis in DLD-1 and HT-29 cells as evidenced by DNA damage, PARP-1 cleavage, Bcl-2 inhibition and phospho-Akt inhibition. This synergistic effect correlated with reactive oxygen species (ROS) production and glutathione (GSH) depletion. NADPH oxidase, mitochondrial complex III and mitochondrial transmembrane potential hyperpolarization contributed toward ROS overproduction when the Li/VPA combination was used. Pretreatment with 4-(2-aminoethyl) benzenesulfonyl fluoride (ABESF; a NADPH oxidase inhibitor) or antimycin A (a complex III inhibitor) partially reversed the apoptotic effect of the Li/VPA, which indicates that ROS from the NADPH oxidase or complex III may play a critical role in the increased proapoptotic effect of Li/VPA. In conclusion, Li enhanced the apoptotic effect of VPA, possibly through an enhanced oxidative stress regulation mechanism. The Li/VPA combination may be a powerful agent against human colon cancer.