Based on the various pharmacological activities of tamarixetin, the present study investigated the cytotoxicity property of tamarixetin in human liver cancer cells including PLC/PRF/5 and HepG2 cells, and their xenografted tumor nude mice. In cells, tamarixetin incubation resulted in the suppression on cell viability; enhanced cell apoptosis rate, LDH release, caspase-3 activation, and reactive oxygen species accumulation; and decreased mitochondrial membrane potential in a dose-dependent manner. Tamarixetin inhibited the growth of PLC/PRF/5- and HepG2-xenografted tumors in BALB/c nude mice after 14-day administration without influencing their bodyweights and organ functions including liver and spleen. Tamarixetin enhanced the expression levels of pro-apoptotic proteins including Bax and cleaved caspase-3 and inhibited the expression levels of anti-apoptotic proteins including Bcl-2 and Bcl-xL in liver cancer cells and their xenografted tumor tissues. Furthermore, tamarixetin significantly suppressed the phosphorylation of ERKs and AKT in both PLC/PRF/5 and HepG2 cells, and tumor tissues. All present data suggest that tamarixetin displays pro-apoptotic properties in liver cancer cells related to the mitochondria apoptotic pathway via regulating the ERKs and AKT signaling.