Recent findings demonstrated proinflammatory functions of interleukin (IL)-9-producing T helper type (Th) 9 cells in the pathogenesis of intestinal bowel diseases (IBDs). However, also antiinflammatory properties have been ascribed to Th9 cells, pointing to a functional heterogeneity. To dissect the specific expression pattern and, especially, diversity of murine antigen-specific Th9 cells, we applied single cell transcription profiling. Th9 cells displayed reduced expression of typical activation markers, such asCd40 ligandandCd96, whereas expression ofCd25andCd83was increased compared with other Th subsets. Importantly, we identified two subsets of Th9 cells differing above all in their CD96 expression. The heterogeneous CD96 expression was specific for Th9 cells and not observed for other Th subtypes, such as Th1 cells. Lower CD96 expression was also observed in human IL-9+compared with IFN-γ+T cells. AlthoughIl9was highly transcribed by all Th9 cells, IL-9 mRNA and protein expression was increased in CD96lowcells. Transfer of CD96lowTh9 cells into recombination activating gene 1-deficient (Rag1 -/- ) mice caused severe weight loss, intestinal and colonic inflammation, and destruction of allogeneic skin grafts and thus showed high inflammatory potential. This was associated with their expansion and tissue accumulation. Contrastingly, CD96highTh9 cells did not cause colitis and showed reduced expansion and migratory potential. Blockade of CD96 completely restored the expansion and inflammatory properties of CD96highTh9 cells. Collectively, our data suggest an inhibitory role for the cosignaling receptor CD96 in Th9 cells, raising new opportunities in the treatment of IL-9-associated inflammations such as IBD.