Tenuigenin (TEN), a major active component of polygala tenuifolia root, has been reported to have a number of biological properties, such as anti-oxidative and anti-inflammatory activities. However, the protective effect of TEN on acute liver injury has not yet been reported. This research aims to detect the protective effect of TEN on lipopolysaccharide (LPS) and d-galactosamine (D-GalN)-induced acute liver injury in mice and to investigate the molecular mechanisms. TEN was administered intraperitoneally 1 h before LPS/D-GalN treatment. The levels of TNF-α, IL-1β, ALT, and AST were measured. The expression of NF-κB, ASK1, MAPKs, Nrf2, and HO-1 were detected by western blot analysis. The results showed that TEN significantly inhibited LPS/D-GalN-induced serum ALT and AST levels. TEN also inhibited LPS/D-GalN-induced TNF-α and IL-1β production. Furthermore, LPS/D-GalN-induced hepatic MDA and MPO activities were also inhibited by TEN. In addition, TEN was found to inhibit LPS/D-GalN-induced ASK1 expression, NF-κB and MAPKs activation and up-regulate the expression of Nrf2 and HO-1. In conclusion, TEN protected against LPS/GalN-induced acute liver injury by suppressing inflammatory and oxidative responses.