Cucurbitacin R is known to exhibit an anti-inflammatory effect in different experimental models of inflammation. In this article, we outline the effect of cucurbitacin R on T lymphocyte proliferation, cytokine production, and nuclear factor activation, as well as its influence on various experimental models of delayed-type hypersensitivity (DTH) in mice. Cucurbitacin R reduced the proliferation of phytohemagglutinin A-stimulated human T lymphocytes (IC₅₀, 18 μM), modifying the cell cycle, as well as the production of cytokines [interleukin (IL)-2, IL-4, IL-10, and especially interferon-γ] and the induction of the principal cyclins implicated in the cell cycle (A₁, B₁, D₂, and E). These effects are brought on by a novel, selective inhibition of nuclear factor AT (NFAT) by cucurbitacin R, with no concomitant effect on other transcription factors such as activator protein-1. In addition, we tested the in vivo effects of cucurbitacin R in three experimental models of DTH, as well as its effects on T lymphocyte proliferation, the cell cycle, cytokines, and cyclins. Although cucurbitacin R was found to reduce the inflammatory response brought on by both oxazolone and dinitrofluorobenzene, its activity was even more pronounced against sheep red blood cell-induced edema in mouse paws, with a clear reduction in the production of IL-1β, IL-4, and tumor necrosis factor α in the inflamed paw. In conclusion, cucurbitacin R has the potential to be a new immunosuppressive agent with antiproliferative effects through the inhibition of the NFAT with anti-inflammatory activity in DTH reactions.