Regulatory CD4+ T cells are important for the homeostasis of the immune system and their absence correlates with autoimmune disorders. Here, we investigate the capacity of IL-27, a cytokine with pro- and anti-inflammatory properties, to regulate the generation of transforming growth factor beta (TGFbeta)-inducible forkhead box P3 (Foxp3)-positive regulatory T (Treg) cells. Our results demonstrate that IL-27 inhibits the acquisition of the Treg phenotype at the level of Foxp3, CD25 and CTLA-4 (CD152) expression as well as the suppressive function. In contrast to TGFbeta-induced Treg cells, the cells generated after differentiation in the presence of TGFbeta and IL-27 maintained the ability for IL-2 and tumour necrosis factor alpha (TNFalpha) production. The inhibitory effect of IL-27 on Treg generation was at least partially signal transducer and activator of transcription 3 (STAT3) dependent as examined by targeted STAT3 protein inhibition using small interfering RNA (siRNA), while STAT1-dependent signals seemed to oppose the STAT3 signals. In turn, TGFbeta blocked IL-27-induced T(h)1 differentiation. Thus, IL-27 and TGFbeta mutually control their effects on CD4+ T-cell differentiation, whereby IL-27 favours inflammatory conditions through a STAT3-dependent inhibition of Treg generation.