Background Interleukin-13 (IL-13) is recognized to be a key modulator in the pathogenesis of Th2-induced allergic inflammation. Transcription factors GATA3 and NFAT1 have been both implicated in the regulation of Th2 cytokines. We previously demonstrated the GATA3-NFAT1 association during human T cell activation. However, the function of the GATA3-NFAT1 complex in Th2 cytokines regulation is still unknown. Small interference RNA (siRNA) was constructed to knock down GATA3 expression in Hut-78 cells to investigate the possible role of GATA3-NFAT1 complex in IL-13 transcription. Methods Cells were stimulated with anti-CD3 plus anti-CD28 antibodies to mimic in vivo antigen-mediated co-stimulation; the expression of IL-13 mRNA was determined by real-time PCR; chromation immunoprecipitation (CHIP) assay was employed to investigate the NFAT1 binding to IL-13 promoter. Results GATA3 siRNA suppressed the expression of GATA3 both in mRNA and protein levels in Hut-78 cells. The binding of NFAT1 to IL-13 promoter was inhibited by GATA3 siRNA in activated T cells, which was followed by the reduction of IL-13 transcription. Conclusion GATA3-NFAT1 complex may play an important role in the regulation of IL-13 transcription in human T cells.