This study examined the effect of genistein from Hydrocotyle sibthorpioides on lipopolysaccharide (LPS)/ D -galactosamine ( D -GalN)-induced acute hepatic failure. Compared to the model control, genistein treatment significantly protected against LPS/ D -GalN-induced liver injury, as evidenced by the decrease in serum alanine and aspartate aminotransferases activities and the attenuation of histopathological changes. Furthermore, genistein alleviated the pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α) and nitric oxide (NO)/inducible nitric oxide synthase (iNOS) by inhibiting nuclear factor-κB (NF-κB) activity. Genistein attenuated the elevated level of caspases-3, while augmented the expression of Bcl-2. In addition, LPS/ D -GalN induced significant increase of heme oxygenase (HO), carbon monoxide and bilirubin levels and these alterations were augmented by genistein treatment. In conclusion, the protective effect of genistein on LPS/ D -GalN-induced liver damage was mainly due to its ability to block NF-κB signaling pathway for anti-inflammation response, attenuate hepatocellular apoptosis and increase HO level. These findings suggest that genistein can be considered as a potential agent for preventing acute hepatic failure.