Neuropsychiatric dysfunction and reactive microglia are hallmarks of high-fat diet (HFD)-induced obesity, yet whether these reactive microglia contribute to HFD-induced obesity-related behavioral abnormalities and the underlying mechanisms remain unclear. Here, we show that HFD feeding causes social deficits and anxiety-like behaviors with impaired neuronal activity and alters the gut microbiota, particularly by depleting Lactobacillus reuteri (L. reuteri), in mice. The profiles of microbiome and metabolome in HFD-fed mice predict that specific microbial taxa and their metabolites regulate HFD-induced obesity-related behavioral abnormalities. Oral treatment with the L. reuteri reduces microglial activation and increases dendritic spine density, thus ameliorates social deficits and anxiety in HFD-fed mice. HFD-fed mice that are administered L. reuteri are also found to accumulate butyrate in their gut, sera and brain. Moreover, supplementation of butyrate improves behavioral abnormalities and modulates microglial homeostasis in HFD-fed mice. In addition, selectively removal of microglia through a pharmacologic approach can rescue dendritic spine loss and increase neuronal activity that profoundly alleviates social deficits and anxiety arising from HFD-induced obesity. Overall, this study reveals an unexpected pivotal role of gut commensal-derived butyrate in HFD-induced social deficits and anxiety-like behaviors through regulation of microglial homeostasis and identifies a potential probiotic treatment for HFD-induced obesity-related behavioral abnormalities.