The HMBOX1 (Homeobox Containing 1) gene was first isolated from the human pancreatic cDNA libraries and is widely expressed in many tissues. Previously, we detected high expression of HMBOX1 in the liver, but its function was unclear. In this study, hepatocyte-specific HMBOX1 knockout mice (Hm▵hep mice) were generated and used to characterize the function of HMBOX1 in the LPS/D-GalN-induced acute liver failure model. HMBOX1-knockout exhibits exacerbated liver injury induced by LPS/D-GalN, accompanied with high levels of inflammatory cytokines both in the liver and in circulation. Further investigation demonstrated that HMBOX1 negatively regulates NF-κB signal transduction. Therefore, HMBOX1-knockout in hepatocytes promotes CCL2 expression through the activation of NF-κB signaling, which enhanced the infiltration of macrophages into the liver. In addition, the decrease of HMBOX1 in hepatocytes promotes the activation of macrophages, upregulating CD80 and MHCII, as well as inflammatory factors TNF-α and IL-6. Importantly, overexpression of HMBOX1 rescued liver injury in Hm▵hep mice. These findings indicate that HMBOX1 in hepatocytes acts as a key immunosuppressive factor for inflammation and plays a critical protective role in LPS/D-GalN-induced liver injury.