Shochu distillation remnants (SDR) are by-products in the manufacturing process of the Japanese liquor Shochu and include various useful organic compounds derived from the fermentation of grains. We have obtained valuable powder (PSDR) from freeze-dried SDR by the treatment with ethanol. In this study, we examined the anticancer effects of barley-, rice-, and sweet potato-PSDR against HepG2 and HuH-7 cells of human hepatocellular carcinoma (HCC) in vitro. All PSDR inhibited the growth of both these HCC cells through the induction of apoptosis. Especially, barley-PSDR was the most effective for the growth inhibition and apoptosis induction of HCC cells of all NOR. We next examined the apoptotic mechanisms induced by barley-PSDR Decrease in mitochondrial membrane potential and release of cytochrome c from mitochondria were observed in HCC cells after the treatment with barley-PSDR Furthermore, barley-PSDR induced the nuclear translocation of apoptosis-inducing factor (AlF) from mitochondria, while it did not significantly affect the activities of caspase-3, -8, and -9. The results suggested that barley-NOR induced apoptosis against HCC cells via the caspase-independent mitochondrial pathway. The findings in this study suggest that PSDR has the possibility of therapeutic and/or preventive agents of HCC. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.