Abstract: To explore the effect of the crude extraction from distiller's grains on the proliferation and apoptosis of human hepatocellular carcinoma cell HepG2, the crude extraction from distiller's grains were analyzed by LC-MS firstly, then used to treat HepG2 cells by different concentrations. RTCA was used to detect its effects on proliferation of HepG2 cells. Flow cytometry was used to detect its effects on the cycle of HepG2 cells. Real-Time qPCR was used to detect its effects on apoptosis of HepG2 cells. Western Blot was used to detect the expression of apoptotic-related protein. 43 substances in the crude extraction from distiller's grains were determined by LC-MS. The proliferation of HepG2 cells were significantly inhibited by the crude extraction from distiller's grains and presented concentration dependent; The crude extraction from distiller's grains significantly down-regulated the expression of mRNA and protein of S phase CyclinA and its kinase CDK2, reduced the expression of mRNA and protein of Bcl-2 and increased the expression of mRNA and protein of Bax (P<0.05); the expression levels of CYTC, Cleaved-Caspase-9 and Cleaved-Caspase-3 were increased gradually (P<0.05), the expression levels of Caspase-9, Caspase-3 were decreased (P<0.05). The results showed that the crude extraction from distiller's grains that contained a variety of active substances can significantly inhibit the proliferation of HepG2 cells and may induce apoptosis of HepG2 cells by activating the apoptotic mitochondrial pathway.