Magnolia species have been used for the treatment of allergic diseases in Asia as folk medicine; however, the cellular and molecular mechanisms of its anti-allergic effects have rarely been investigated. In this study, we demonstrated that a methanolic extract of the fructus of Magnolia denudata has suppressive effects on Th2 cytokine production such as IL-4 and IL-13, but not IFN-γ and IL-17, produced by both phorbol 12-myristate 13-acetate/ionomycin (PI)- and CD3/CD28-stimulated EL-4 T cells. Moreover, the mRNA expression of Th2 cytokines was significantly inhibited, and luciferase activity in cells transiently transfected with IL-4 or IL-13 promoter reporter plasmids was suppressed by M. denudata, indicating that M. denudata may regulate these expression at the transcriptional level. Western blot analysis for transcription factors involved in the cytokine gene expression indicated that the activation of c-Jun was significantly downregulated in the nucleus of cells, while the activations of nuclear factor of activated T cells, nuclear factor kappa B and c-Fos, were not affected. Furthermore, the mRNA expression and nuclear translocation of GATA-binding protein 3, a key transcriptional factor for Th2 commitment and Th2 cytokine expression, but not T-bet and RORγt, were dramatically downregulated by M. denudata. Treatment with M. denudata suppressed the phosphorylation of p38 mitogen-activated protein kinase; however, the PI-induced phosphorylation of extracellular signal-related kinase and c-Jun N-terminal kinase was unaffected. Taken together, our study indicated that M. denudata inhibited IL-4 and IL-13 expression, possibly through regulation of p38 mitogen-activated protein kinase phosphorylation and selective transcription factors, such as GATA-3 and c-Jun, in EL-4 T cells.