The human gut microbiota ferments carbohydrates to produce metabolites that mediate beneficial community functions. While the production of the short-chain fatty acids (SCFAs) acetate, propionate, and butyrate is well characterized, how valerate and caproate are produced has remained enigmatic.Here, we characterize the production of valerate and caproate by the gut microbiota. In a continuous culture model of a defined gut bacterial community, we observed that valerate took longer to reach physiological levels than acetate, propionate, or butyrate and correlated with Lachnospiraceae. This led us to test valerate and caproate production by members of Lachnospiraceae. While tested Lachnospiraceae isolates produced low levels of valerate and caproate at baseline, physiological concentrations of SCFAs activated valerate and caproate production in a species-specific manner. Furthermore, providing isotopically labelled SCFAs revealed that isolates of Anaerostipes hadrus and Coprococcus comes, which are members of Lachnospiraceae, directly incorporate exogenous acetate, propionate, and butyrate into valerate and caproate in a manner consistent with the reverse β-oxidation pathway. Therefore, we examined this pathway in Lachnospiraceae and found that Lachnospiraceae spp. encode different copy numbers and variants of the enzyme acetyl-CoA acetyltransferase (ThlA). ThlA performs the first step in reverse β-oxidation by condensing acyl-CoA molecules. We demonstrate that tested Lachnospiraceae ThlA protein variants consume propionyl-CoA and acetyl-CoA substrates consistent with valerate production. Coprococcus comes, the only tested species to produce caproate, encodes a second ThlA protein with increased activity on butyryl-CoA. In human fecal samples, multiple genera of Lachnospiraceae correlate with butyrate, while Coprococcus also correlates with caproate levels.These findings reveal a role of certain members of the Lachnospiraceae as prominent producers of valerate and caproate within human microbiomes through the reverse β-oxidation pathway.