We investigated the protective effects exerted by oridonin, the main active constituent of the Chinese medicinal herb Rabdosiarubescens, against lipopolysaccharide (LPS)/D-galactosamine (D-Gal)-induced acute liver injury (ALI). An ALI model was induced in mice using LPS (40 mu g/0.5 ml) and D-Gal (5 mg/0.5 ml). The mice were randomly divided into the following five groups of six mice each: one control group (a), one ALI group (b), two oridonin treatment groups (c and d), and one oridonin control group (e). Oridonin (0.2 mg/0.5 ml) was administered once 1 h prior to the LPS/D-Gal challenge in group c and a total of three times over a period of four days, with the last dose given at 1 h before the LPS/D-Gal challenge, in group d. Pretreatment with oridonin improved the survival rate, alleviated histopathological abnormalities, and suppressed plasma aminotransferases in the LPS/D-Gal-challenged mice. Importantly, oridonin attenuated LPS/D-Gal-induced apoptosis in hepatocytes by reducing pro-apoptotic signals (P<0.05), such as tumor necrosis factor-alpha (TNF-alpha) and c-Jun N-terminal kinases (JNK). Furthermore, JNK-associated mitochondrial pro-apoptotic proteins were also suppressed by pretreatment with oridonin. Taken together, these data show that oridonin exerts protective effects against LPS/D-Gal-induced ALI in mice via a mechanism that may involve the suppression of the pro-apoptotic cytokine TNF-alpha and JNK-associated pro-apoptotic signaling.