Growth arrest and DNA damage inducible protein 34 (GADD34) is induced by various cellular stresses, such as DNA damage, endoplasmic reticulum stress, and amino-acid deprivation. Although the major roles of GADD34 are regulating ER stress responses and apoptosis, a recent study suggested that GADD34 is linked to innate immune responses. In this report, we investigated the roles of GADD34 in inflammatory responses against bacterial infection. To explore the effects of GADD34 on systemic inflammation in vivo, we employed a lipopolysaccharide (LPS)-induced murine sepsis model and assessed the lethality, serum cytokine levels, and tissue injury in the presence or absence of GADD34. We found that GADD34 deficiency increased the lethality and serum cytokine levels in LPS-induced sepsis. Moreover, GADD34 deficiency enhanced tissue destruction, cell death, and pro-inflammatory cytokine expression in LPS-induced acute liver injury. Pro-inflammatory cytokine production after LPS stimulation is regulated by the Toll-like receptor 4 (TLR4)-mediated NF-κB signaling pathway. In vitro experiments revealed that GADD34 suppressed pro-inflammatory cytokine production by macrophages through dephosphorylation of IKKβ. In conclusion, GADD34 attenuates LPS-induced sepsis and acute tissue injury through suppressing macrophage activation. Targeting this anti-inflammatory role of GADD34 may be a promising area for the development of therapeutic agents to regulate inflammatory disorders.