RING finger protein186 (RNF186) is dramatically upregulated in steatotic livers. The physiological role of RNF186 in non-alcoholic fatty liver disease (NAFLD) remains obscure. Here, we found that hepatocyte-specific RNF186 knockout (RNF186 LKO ) mice were protected from HFD-induced obesity. RNF186 ablation in liver suppressed inflammatory responses and ER stress and alleviated insulin tolerance, leading to improved glucose and lipid metabolism under HFD conditions. RNA-seq and western blot analyses revealed a significant downregulation of peroxisome proliferator-activated receptor γ, stearoyl-CoA desaturase 1, and cluster of differentiation 36 in the liver of RNF186 knockout mice consuming HFD. RNF186 deletion in liver results in less weight gain during HFD feeding and is associated with reduced liver fat, inflammation, and improved glucose and insulin tolerance. In contrast, upregulation of RNF186 in C57BL/6J mice livers impaired lipid metabolism and insulin tolerance. The collective results suggest that RNF186 may be a potential regulator of NAFLD in obesity.