Sepsis is the leading cause of death for intensive care patients. Lipopolysaccharide (LPS) administration to animals under anesthesia is a strategy for the study of uncontrolled release of proinflammatory cytokines. Anesthetics have been indicated that they can specially affect immune responses, such as the inflammatory response. Pentobarbital is an anesthetic used mainly in animal studies. Thus, the effect of pentobarbital on tumor necrosis factor-α (TNF-α) release was determined. The results revealed that pentobarbital suppressed the expression of TNF-α mRNA and its proteins, which may result from the decrease in the activities of nuclear factor-κB and activator protein 1 and the reduction of the expression of p38 mitogen-activated protein kinase by pentobarbital. After the inhibitory activity of the pentobarbital for TNF-α release was proven in vivo, the cytotoxic effects of LPS were examined in vivo with or without pentobarbital treatments. In vivo results indicated that plasma levels of alanine aminotransferase, aspartate aminotransferase, lactic dehydrogenase, creatine kinase, serum urea nitrogen, and amylase decreased dramatically in the anesthetic group with pentobarbital administration. Finally, the effect of pentobarbital on TNF-α-related cell death was monitored in vitro, and the results indicated that pentobarbital could directly enhance the viabilities of cells under the treatment of TNF-α and protected cells from apoptosis induced by deferoxamine mesylate-induced hypoxia. These results suggest that pentobarbital significantly influences the LPS-induced inflammatory responses and protects cells from death directly and indirectly induced by TNF-α. The information provides a perspective to re-evaluate the results of the experiments in which animals were anesthetized with pentobarbital. The anti-inflammatory effects of the drugs may have been caused by the synergistic effect of pentobarbital.