Ischemia/reperfusion (I/R) injury constitutes a major reason for failure of liver surgeries and transplantation. I/R injury is more severe in steatotic livers and limits their use in transplantation. Here, we present a novel and selective Toll-like receptor 9 (TLR9) antagonist COV08-0064 and test its potential to protect from I/R-induced injury in normal and steatotic livers. The in vivo effects of COV08-0064 pretreatment were investigated on normal chow diet (NCD) and high fat diet (HFD)-fed mice subjected to segmental (70%) warm hepatic I/R. Also, the in vitro effects of COV08-0064 were elucidated in murine macrophages and dendritic cells. Mice on a HFD had pronouncedly greater hepatic I/R injury than mice on a NCD. COV08-0064-pretreatment to both NCD and HFD-fed mice reduced hepatic I/R injury. COV08-0064-pretreatment was associated with less production of the liver inflammatory cytokines and mediators TNF-α, IL-1β, IL-6, NLRP3, iNOS and MCP-1. These manifestations were preceded with inhibition of JNK and ERK phosphorylation and TLR9 cleavage in the liver. COV08-0064 enhanced the hepatic expression of the endogenous anti-inflammatory cytokines IL-10 and IL-1Ra at the early phase I/R injury. In vitro, COV08-0064 selectively blocked mRNA upregulation of TNF-α, IL-1β, NLRP3 and MCP-1 in macrophages and IFN-β mRNA in dendritic cells induced by the TLR9 agonist CpG-ODN. These effects were concordant with inhibition of JNK, ERK, IκBα and IKKα/β phosphorylation. In conclusion, TLR9 signaling inhibition by COV08-0064 may be an effective approach in liver surgeries including transplantation to limit I/R-injury and overcome the shortages in the donor pool by incorporating steatotic livers.