Schisandrin B is an active component isolated from Schisandra chinensis (TurcZ.) Baill. that is widely used as an antihepatotoxic agent. Schisandrin B has significant hepatoprotective effect against chemical and immunological liver injury. This study aimed to investigate the effect of Schisandrin B on the expression of 27- and 70-kDa heat-shock protein (HSP) and its role in protection against acetaminophen-induced liver injury in mice. After the mice were pretreated, Western blot and real-time quantitative polymerase chain reaction were used to detect the protein and gene expression of HSP27 and HSP70, respectively; the liver tissues were subjected to histological evaluation, and alanine aminotransferase and aspartate aminotransferase activities in the serum were measured. Oral administration of Schisandrin B increased the expression of HSP27 and HSP70 in a time- and dose-dependent manner. The inducing effect of Schisandrin B on HSP27 and HSP70 was also confirmed by real-time quantitative polymerase chain reaction. In the acetaminophen-induced liver injury mouse model, the prior oral administration of Schisandrin B (200 mg/kg) three times in 24 h markedly alleviated liver injury as indicated by the amelioration of histopathological hepatic necrosis and the reduction of alanine aminotransferase and aspartate aminotransferase activities in the serum. However, the earlier actions of Schisandrin B were all suppressed significantly by Quercetin, a known HSP inhibitor. The hepatic cytoprotective action of Schisandrin B against acetaminophen-induced liver injury is mediated, at least in part, by the induction of HSP27 and HSP70 in mice.