We previously prepared a standardized and purified extract of Salvia miltiorrhiza, PF2401-SF, and showed that it protected against hepatic injury more effectively than ethanol based extraction. In this study, we determined the hepatoprotective mechanisms of PF2401-SF in vivo. Hepatic injury was induced in mice by using carbon tetrachloride (CCl4). Treatment with PF2401-SF (1 or 10 mg/kg, p.o.) significantly reduced the levels of alanine transaminase (ALT) and aspartate transaminase (AST) in the plasma. PF2401-SF treatment resulted in further elevation of the CCl4-induced heme oxygenase-1 (HO-1) expression, which contributed to the PF2401-SF-mediated liver protection. Additionally, PF2401-SF treatment significantly reduced the c-Jun NH2-terminal kinase (JNK) phosphorylation induced by CCl4. Taken together, these results suggest that the protective effect of PF2401-SF, a standardized fraction of S. miltiorrhiza, against CCl4-induced hepatic injury in mice arises from its induction effect on HO-1 and inhibitory effect on JNK phosphorylation.