Stephanthraniline A (STA) isolated from the stems of Stephanotis mucronata (Blanco) Merr. was evaluated for their suppression on T cells' immune responses in vitro and in vivo. In vivo, oral administration of STA significantly inhibited T cell-mediated delayed-type hypersensitivity (DTH) response. In vitro, STA has inhibitory effects on T cell proliferation induced by CD3/CD28 cross-linking or Con A; additionally, CD4(+) T cells are more sensitive to this inhibition than CD8(+) T cells. STA also suppressed the production of cytokines (IL-2, IFN-γ, IL-4 and IL-17) and mRNA expression of the genes associated with T cell activation, proliferation and differentiation. Our data indicate that STA inhibits the proliferation of T cells by inducing cell cycle arrest but not inducing apoptosis. The inhibitory mechanism of STA on T cells was correlated with the gene change related to multi-signal transduction pathways. Furthermore, we also provided lines of evidence that STA, distinct from glucocorticoids, did not activate the glucocorticoid receptor. These findings would be beneficial for further understanding the therapeutic effects of S. mucronata in the treatment of autoimmune diseases. It also suggested the potential of the natural steroid STA as the effective candidate compounds for use in the treatment of inflammatory and autoimmune diseases.