Interleukin-27 (IL-27) is an immunoregulatory cytokine that suppresses inflammation through multiple mechanisms, including induction of IL-10, but the transcriptional network mediating its diverse functions remains unclear. Combining temporal RNA profiling with computational algorithms, we predict 79 transcription factors induced by IL-27 in T cells. We validate 11 known and discover 5 positive (Cebpb, Fosl2, Tbx21, Hlx, and Atf3) and 2 negative (Irf9 and Irf8) Il10 regulators, generating an experimentally refined regulatory network for Il10. We report two central regulators, Prdm1 and Maf, that cooperatively drive the expression of signature genes induced by IL-27 in type 1 regulatory T cells, mediate IL-10 expression in all T helper cells, and determine the regulatory phenotype of colonic Foxp3+ regulatory T cells. Prdm1/Maf double-knockout mice develop spontaneous colitis, phenocopying ll10-deficient mice. Our work provides insights into IL-27-driven transcriptional networks and identifies two shared Il10 regulators that orchestrate immunoregulatory programs across T helper cell subsets. Display omitted •IL-27-driven transcriptional network in CD4 T cells unravels key Il10 regulators•Systematic characterization of the function of 16 Il10 regulators by RNA-seq•Identification of transcription factors associated with Il10 in multiple T cell subsets•Prdm1 and Maf are critical for Il10 production and intestinal immune homeostasis Zhang et al. construct a transcriptional network for IL-27-mediated Il10 production in CD4 T cells, characterize the function of 16 Il10 regulators, and uncover the role of two transcription factors, Prdm1 and Maf, in driving Il10 production in all T helper cells and in maintaining immune homeostasis in the colon.