Bile acids are abundant in the mammalian gut, where they undergo bacteria-mediated transformation to generate a large pool of bioactive molecules. Although bile acids are known to affect host metabolism, cancer progression and innate immunity, it is unknown whether they affect adaptive immune cells such as T helper cells that express IL-17a (T H 17 cells) or regulatory T cells (T reg cells). Here we screen a library of bile acid metabolites and identify two distinct derivatives of lithocholic acid (LCA), 3-oxoLCA and isoalloLCA, as T cell regulators in mice. 3-OxoLCA inhibited the differentiation of T H 17 cells by directly binding to the key transcription factor retinoid-related orphan receptor-γt (RORγt) and isoalloLCA increased the differentiation of T reg cells through the production of mitochondrial reactive oxygen species (mitoROS), which led to increased expression of FOXP3. The isoalloLCA-mediated enhancement of T reg cell differentiation required an intronic Foxp3 enhancer, the conserved noncoding sequence (CNS) 3; this represents a mode of action distinct from that of previously identified metabolites that increase T reg cell differentiation, which require CNS1. The administration of 3-oxoLCA and isoalloLCA to mice reduced T H 17 cell differentiation and increased T reg cell differentiation, respectively, in the intestinal lamina propria. Our data suggest mechanisms through which bile acid metabolites control host immune responses, by directly modulating the balance of T H 17 and T reg cells.