The objective of this study was to reveal the exact role of Kupffer cells in the diet-induced insulin resistance, inflammation and liver autophagy. C57BL/6j male mice were fed with either chow diet or high-fat diet (HFD) for 12 weeks. Meanwhile, HFD feeding mice received an intraperitoneal injection of either 0.2% GdCl3 solution (20mg/kg) twice a week to deplete Kupffer cells or natural saline (5mL/kg) as control. The mRNA expressions of Kupffer cells markers (CD68 and F4/80), insulin sensitivity, TNF-α concentration and NF-κB activation and parameters of autophagy were assessed. Results demonstrated that CD68 and F4/80 mRNA expressions in the liver were up-regulated in HFD fed animals, while significantly reduced after GdCl3 administration. HFD feeding led to insulin resistance and TNF-α level and activation of NF-κB in insulin-sensitive tissues (liver, adipose tissue and skeletal muscle) were significantly elevated. Interestingly, alterations above were reversed by varying degrees but significantly after Kupffer cells depletion. Furthermore, western blot showed hepatic LC3-II as well as phosphorylation of AMPK in liver and skeletal muscle were significantly lower in mice fed HFD, and these changes dramatically ameliorated by GdCl3 treating. In conclusion, selective depletion of Kupffer cells significantly attenuated diet-induced insulin resistance, inflammation and promoted liver autophagy. Strategies targeting Kupffer cells function or autophagic processes could be a promising approach to counteract diet induced obesity and related metabolic disorders.