Sestrins (Sesns) are conserved antioxidant proteins that accumulate in cells in response to various stresses. However, the regulatory roles of Sesn2 in the immune system and in inflammatory responses remain obscure. In the present study, we investigated whether Sesn2 regulates Toll like receptor (TLR)-mediated inflammatory signaling and sought to identify the molecular mechanism responsible. In cells expressing Sesn2, it was found that Sesn2 almost completely inhibited lipopolysaccharide (LPS)-induced NO release and iNOS expression. A gene knockdown experiment confirmed the role of Sesn2 in LPS-activated RAW264.7 cells. Consistently, proinflammatory cytokine (e.g., TNF-α, IL-6, and IL-1β) release and expression were inhibited in Sesn2-expressing cells. Furthermore, Sesn2 prevented LPS-elicited cell death and ROS production via inhibition of NADPH oxidase. NF-κB and AP-1 are redox-sensitive transcription factors that regulate the expressions of diverse inflammatory genes. Surprisingly, Sesn2 specifically inhibited AP-1 luciferase activity and its DNA binding, but not those of NF-κB. AP-1 inhibition by Sesn2 was found to be due to a lack of JNK, p38, and c-Jun phosphorylation. Next, we investigated whether Sesn2 protects galactosamine (Gal)/LPS-induced liver injury in mice infected with a recombinant adenovirus Sesn2 (Ad-Sesn2). Ad-Sesn2 present less severe hepatic injury as supported by decreases in the ALT, AST, and hepatocyte degeneration. Moreover, Ad-Sesn2 attenuated Gal/LPS-induced proinflammatory gene expression in mice. The study shows that Sesn2 inhibits TLR-induced proinflammatory signaling and protects cells by inhibiting JNK- or p38-mediated c-Jun phosphorylation.