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Background/Aims: Sepsis and lipopolysaccharides (LPS) cause mild to severe hepatic dysfunction. In this study, Kupffer cell activation, involvement of TNFα and caspases downstream of the TNFα receptor were examined in hepatocyte apoptosis induced by LPS. Methods: In In vivo experiments, male Sprague-Dawley rats were injected intravenously with LPS, and small amounts of the blood and liver were sampled to evaluate apoptosis. Kupffer cells were inactivated by pretreatment with gadolinium chloride for 2 days. In in vitro experiments, hepatocytes and Kupffer cells were separately isolated from rat livers using collagenase perfusion. Results: LPS induced time-dependent and dose-dependentincreases in the number of TUNEL-positive cells, which coincided with the apoptotic features of hepatocytes demonstrated by electron microscopy and DNA ladder. Activation of caspase-3-like proteases was observed with an increase in the number of apoptotic hepatocytes. Immunostaining with activated caspase-3-specific antibody showed that caspase-3 was activated only in the cytoplasm of TUNEL-positive hepatocytes. Inactivation of Kupffer cells by gadolinium chloride was concomitantly accompanied by the prevention of caspase-3 activation, hepatocyte apoptosis and liver injury induced by LPS. The coculture system of hepatocytes and Kupffer cells, but neither cell culture system, individually, showed LPS-induced hepatocyte apoptosis. Kupffer cell-conditioned medium induced hepatocyte apoptosis, whereas addition of anti-TNFα antibody to Kupffer cell-conditioned medium did not. Additions of acetyl-DEVD-CHO, acetyl-YVAD-CHO, and acetyl-IETD-CHO to Kupffer cell-conditioned medium decreased the number of apoptotic hepatocytes. Conclusions: These results suggest that the activation of Kupffer cells, TNFα and caspases downstream of TNFR1 were involved in hepatocyte apoptosis induced by LPS.