T cells are major providers of proinflammatory cytokines. They are preprogrammed in the mouse thymus into distinct subsets producing either interleukin-17 (IL-17) or interferon- (IFN-), which segregate with CD27 expression. In the periphery, CD27 − (27 − ) T cells can be induced under inflammatory conditions to coexpress IL-17 and IFN-; the molecular basis of this functional plasticity remains to be determined. On the basis of differential microRNA (miRNA) expression analysis and modulation in T cell subsets, we identified miR-146a as a thymically imprinted post-transcriptional brake to limit IFN- expression in 27 − T cells in vitro and in vivo. On the basis of biochemical purification of Argonaute 2–bound miR-146a targets, we identified Nod1 to be a relevant mRNA target that regulates T cell plasticity. In line with this, Nod1-deficient mice lacked multifunctional IL-17 + IFN- + 27 − cells and were more susceptible to Listeria monocytogenes infection. Our studies establish the miR-146a/NOD1 axis as a key determinant of T cell effector functions and plasticity.