Chronic immune thrombocytopenia (cITP) is an autoimmune disease with disturbed cytokine profile. Although plasma levels of IL-27 are shown to be associated with cITP, its association with T cell subsets has not been studied. The objective of this study was to study the association between IL-27 and different T cell subsets in patients with cITP. Heparinized blood was collected from 31 patients with cITP and 36 healthy controls (platelet count <100 × 109/l and 103–280 × 109/l, respectively). The percentage of Th1, Th2 and Th17 cells in peripheral blood mononuclear cells (PBMCs) were enumerated by flow cytometry, and the mRNA levels of IL-27, T-bet, GATA-3 and retinoid-related orphan receptor gamma (RORγt) by real-time reverse transcriptase polymerase chain (RT-PCR). Plasma cytokine levels of IL-27, interferon-gamma (IFN-γ), IL-4 and IL-17A were estimated by flow cytometrix. The effect of exogenous recombinant IL-27(rhIL-27) on the differentiation of T cells into Th1, Th2 and Th17 cells was investigated by cell culture. The percentage of Th1 and Th17 cells and the plasma concentration and mRNA levels of IL-27 were significantly higher in cITP patients compared with healthy controls. Plasma levels of IL-27 correlated positively with percentage of Th1 cells in patients with cITP. Exogenous (rhIL-27) could significantly up-regulate the percentage of Th1 cells and down-regulate Th2 cells in vitro. Th17 cells were reduced in the presence of (rhIL-27) in controls but had no effect in patients with cITP. The up-regulation of IL-27 might cause Th1 differentiation and might be involved in the pathophysiology of cITP.