Polymethoxyflavones (PMFs) are flavone compounds that contain more than two methoxyl groups and are almost exclusively found in citrus peel. In this study, we examined and compared the effects of the PMFs nobiletin, heptamethoxyflavone and natsudaidain on antigen-specific T cell activation in vitro and in vivo. The three PMFs suppressed proliferation responses in an in vitro study. The PMFs also suppressed IFN-γ, IL-2 and IL-10 production from splenocytes in a dose-dependent manner. Interestingly, nobiletin and heptamethoxyflavone, but not natsudaidain, enhanced IL-4 production from splenocytes. In an in vivo model, antigen-specific T cell proliferation responses were reduced in mice treated with nobiletin, heptamethoxyflavone and natsudaidain compared with responses in control mice. The results suggest that citrus PMFs suppress T cell activation and that nobiletin and heptamethoxyflavone enhance IL-4 production.