The gut microbiota plays a key role in host metabolism. Toll-like receptor 5 (TLR5), a flagellin receptor, is required for gut microbiota homeostasis. Accordingly, TLR5-deficient (T5KO) mice are prone to develop microbiota-dependent metabolic syndrome. Here we observed that T5KO mice display elevated neutral lipids with a compositional increase of oleate C18:1 (n9) relative to wild-type littermates. Increased oleate contribution to hepatic lipids and liver SCD1 expression were both microbiota dependent. Analysis of short-chain fatty acids (SCFAs) and 13C-acetate label incorporation revealed elevated SCFA in ceca and hepatic portal blood and increased liver de novo lipogenesis in T5KO mice. Dietary SCFAs further aggravated metabolic syndrome in T5KO mice. Deletion of hepatic SCD1 not only prevented hepatic neutral lipid oleate enrichment but also ameliorated metabolic syndrome in T5KO mice. Collectively, these results underscore the key role of the gut microbiota-liver axis in the pathogenesis of metabolic diseases. Display omitted •T5KO mice microbiota generates more cecal SCFA, substrates for hepatic lipogenesis•Dietary SCFA aggravate metabolic syndrome in T5KO mice•Hepatic SCD1 plays a key role in the development of metabolic syndrome in T5KO mice•Metabolic syndrome in T5KO mice is microbiota-liver axis dependent Singh et al. identify a gut microbiota-liver axis responsible for the metabolic syndrome developed by TLR5-deficient mice and show that short-chain fatty acids generated by gut bacterial fermentation of dietary fiber fuel SCD1-mediated lipogenesis in the liver, which promotes insulin resistance and inflammation.