The immune and enteric nervous (ENS) systems monitor the frontier with commensal and pathogenic microbes in the colon. We investigated whether FoxP3+ regulatory T (Treg) cells functionally interact with the ENS. Indeed, microbe-responsive RORγ+ and Helios+ subsets localized in close apposition to nitrergic and peptidergic nerve fibers in the colon lamina propria (LP). Enteric neurons inhibited in vitro Treg (iTreg) differentiation in a cell-contact-independent manner. A screen of neuron-secreted factors revealed a role for interleukin-6 (IL-6) in modulating iTreg formation and their RORγ+ proportion. Colonization of germfree mice with commensals, especially RORγ+ Treg inducers, broadly diminished colon neuronal density. Closing the triangle, conditional ablation of IL-6 in neurons increased total Treg cells but decreased the RORγ+ subset, as did depletion of two ENS neurotransmitters. Our findings suggest a regulatory circuit wherein microbial signals condition neuronal density and activation, thus tuning Treg cell generation and immunological tolerance in the gut. Display omitted •Treg cells in the colon lamina propria reside close to neuron projections•Neurons modulate the differentiation and phenotype of iTreg cells in culture via IL-6•Neuron-specific ablation of Il6 increases the number of RORγ+ Treg cells in vivo•Microbial colonization affects a subset of neurons in the enteric nervous system Regulatory T (Treg) cells lie in proximity to nerve fibers in the colon lamina propria. Yan et al. reveal a regulatory circuit wherein microbial signals condition neuronal density and activation, which in turn, via neuron-produced IL-6, tunes Treg cell generation, which has implications for intestinal tolerance.