Piperine, a pungent alkaloid found in the fruits of black pepper plants, has diverse physiological effects, including the ability to inhibit immune cell-mediated inflammation. Since the cytokine interleukin-2 (IL-2) is essential for the clonal expansion and differentiation of T lymphocytes, we investigated the effect of piperine on IL-2 signaling in IL-2-dependent mouse CTLL-2 T lymphocytes. Tritiated-thymidine incorporation assays and flow cytometric analysis of Oregon Green 488-stained cells showed that piperine inhibited IL-2-driven T lymphocyte proliferation; however, piperine did not cause T lymphocytes to die or decrease their expression of the high affinity IL-2 receptor, as determined by flow cytometry. Western blot analysis showed that piperine blocked the IL-2-induced phosphorylation of signal transducer and activator of transcription (STAT) 3 and STAT5 without affecting the upstream phosphorylation of Janus kinase (JAK) 1 and JAK3. In addition, piperine inhibited the IL-2-induced phosphorylation of extracellular signal-regulated kinase 1/2 and Akt, which are signaling molecules that regulate cell cycle progression. Piperine also suppressed the expression of cyclin-dependent kinase (Cdk) 1, Cdk4, Cdk6, cyclin B, cyclin D2, and Cdc25c protein phosphatase by IL-2-stimulated T lymphocytes, indicating G0/G1 and G2/M cell cycle arrest. Piperine-mediated inhibition of IL-2 signaling and cell cycle progression in CTLL-2 T lymphocytes suggests that piperine should be further investigated in animal models as a possible natural source treatment for T lymphocyte-mediated transplant rejection and autoimmune disease.