Tumor necrosis factor-α (TNF-α) contributes to liver injury by inducing hepatocyte apoptosis. Recent evidence suggests that cathepsin B (cat B) contributes to TNF-α-induced apoptosis in vitro. The aim of the present study was to determine whether cat B contributes to TNF-α-induced hepatocyte apoptosis and liver injury in vivo. Cat B knockout ( catB −/−) and wild-type ( catB +/+) mice were first infected with the adenovirus Ad5IκB expressing the IκB superrepressor to inhibit nuclear factor-κB-induced survival signals and then treated with murine recombinant TNF-α. Massive hepatocyte apoptosis with mitochondrial release of cytochrome c and activation of caspases 9 and 3 was detected in catB +/+mice 2 hours after the injection of TNF-α. In contrast, significantly less hepatocyte apoptosis and no detectable release of cytochrome c or caspase activation occurred in the livers of catB −/− mice. By 4 hours after TNF-α injection, only 20% of the catB +/+ mice were alive as compared to 85% of catB −/− mice. Pharmacological inhibition of cat B in catB +/+ mice with l-3-trans-(propylcarbamoyl)oxirane-2-carbonyl- l-isoleucyl- l-proline (CA-074 Me) also reduced TNF-α-induced liver damage. The present data demonstrate that a cat B-mitochondrial apoptotic pathway plays a pivotal role in TNF-α-induced hepatocyte apoptosis and liver injury.