In this study, we found that pretreatment with low dose of lipopolysaccharide (LPS), also known as lipoglycans and endotoxin, obviously attenuated liver injury caused by diethylnitrosamine (DEN) in mice. This protective effect was described by decreased ALT, TNF-α, and IL-1β and increased TGF-β production. However, Toll-like receptor 4-deficient (TLR4(-/-)) or macrophages depletion abolished this protection in mice, which revealed Kupffer cells (KCs) and TLR4 to be crucial for the prevention of LPS against DEN-induced damage. Further study revealed that LPS pretreatment induced the KCs to M2 polarization and impaired the signaling of MAPKs and NF-κB that mediated the production of inflammatory cytokines. Moreover, T regulatory cells (Tregs) were also recruited to the liver, which may mediate immunosuppression and participate in the prevention of DEN-induced injury. Our results suggested that LPS protected against DEN-induced hepatitis via induction of M2 Kupffer cells and recruitment of Tregs, which contributes to liver tolerance in TLR4-dependent mechanism.