Th9 cells protect hosts against helminthic infection but also mediate allergic disease. Here we show that nitric oxide (NO) promotes Th9 cell polarization of murine and human CD4(+) T cells. NO de-represses the tumour suppressor gene p53 via nitrosylation of Mdm2. NO also increases p53-mediated IL-2 production, STAT5 phosphorylation and IRF4 expression, all essential for Th9 polarization. NO also increases the expression of TGFβR and IL-4R, pivotal to Th9 polarization. OVA-sensitized mice treated with an NO donor developed more severe airway inflammation. Transferred Th9 cells induced airway inflammation, which was exacerbated by NO and blocked by anti-IL-9 antibody. Nos2(-/-) mice had less Th9 cells and developed attenuated eosinophilia during OVA-induced airway inflammation compared with wild-type mice. Our data demonstrate that NO is an important endogenous inducer of Th9 cells and provide a hitherto unrecognized mechanism for NO-mediated airway inflammation via the expansion of Th9 cells.