Rho kinase (ROCK) inhibition has been reported to improve various inflammatory diseases including endotoxemia. Mitochondrial dysfunction might be the key to the pathophysiology of sepsis-induced organ failure. Therefore, this study aimed to explore whether ROCK inhibition protects against the liver injury by regulating mitochondrial function in endotoxemia model mice. The mice were randomly divided into four groups (N = 6–8 per group): control, LPS, LPS + Y-27632 (LPS + Y), and LPS + Mito-TEMPO (LPS + M). For induction of endotoxin-induced acute liver injury, the mice were intraperitoneally administered lipopolysaccharide (LPS, 20 mg/kg). The ROCK inhibitor Y-27632 (or mitochondrial antioxidant Mito-TEMPO) was intraperitoneally administered at 18 and 1 h before injection of LPS. The mice were euthanized 8 h after LPS administration. The liver and blood samples were taken and preserved for analysis. Results of this study showed that pretreatment with Y-27632 or Mito-TEMPO significantly attenuated the liver injury as compared to the LPS group. This was confirmed by decreased plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and by reduced hepatocellular apoptosis and histologic damage. Pretreatment with Y-27632 or Mito-TEMPO markedly reduced the LPS-induced inflammatory response and oxidative stress the in liver. Furthermore, it showed similar protective effects on the hepatic mitochondrial function, including an increased activity of complexes I and IV and mitochondrial superoxide dismutase (MnSOD), and an upregulated expression of mtDNA-encoded genes. Taken together, these data demonstrate that Mito-TEMPO can potently inhibit the endotoxin-induced mitochondrial and hepatic abnormalities and indicate that mitochondrial dysfunction might play a key role in the endotoxemia-induced acute liver injury. Moreover, our study shows that ROCK inhibition protects against the endotoxemia-induced liver injury by improving the mitochondrial function.