Abstract: Background and Objective: Stephanthraniline A (STA), a natural C21 steroid isolated from Stephanotis mucronata (Blanco) Merr., is a potential immunosuppressant. The purpose of this study was to investigate the synergetic effects of STA and cyclosporine A (CsA) on T-cell response. Materials and Methods: First, the synergetic effects of STA and CsA were investigated in vitro on the cell proliferation, CD25 surface expression and cytokine IL-2 production of concanavalin A (Con A)-induced T-cells by MTT method, flow cytometric analysis and ELISA, respectively. Furthermore, 2,4-dinitrofluorobenzene (DNFB)-induced delayed-type hypersensitivity (DTH), a T-cell-mediated response in mice was used to evaluate the synergetic effects of STA and CsA in vivo. Finally, the direct effect of STA on CYP3A4, which is the predominant enzyme for metabolism of CsA was determined using P450-Glo™ CYP3A4 screening system. Results: The results showed that Con A-induced T-cells proliferation, IL-2 production and CD25 expression were not inhibited by low-dose STA (1 μM) or CsA (1 nM) alone but was significantly reduced by the combination of STA and CsA. The DNFB-induced mice ear swelling, hyperplasia and infiltration of inflammatory cells were also significantly diminished by the combined treatment with non-therapeutic dose of STA (1 mg kg–1) and CsA (0.5 mg kg–1). In addition, STA at the concentration of more than 0.1 μM significantly decreased CYP3A4 activity. Conclusion: The STA synergized the inhibitory effects of CsA on T-cell response in vitro and in vivo. These effects were attributable to its different and complementary molecular mechanisms and may partly due to its increasing of the bioavailability of CsA via inhibiting CYP3A4.