Lck is a T cell-restricted Src family protein tyrosine kinase that plays pivotal roles in TCR-mediated signaling. We aimed to identify novel agents that could disrupt the molecular interaction of the Src homology 2-domain of Lck (Lck SH2) with its binding partners, with the expectation that this would impair TCR signaling and generate immunosuppression. Large-scale screening of plant extracts indicated that rosmarinic acid (RosA) in extracts of Prunella vulgaris consistently inhibits the interaction between Lck SH2 and a peptide containing its consensus binding sequence (pYEEI). The inhibitory effect of RosA was specific for SH2 domains of Src family protein tyrosine kinase. RosA inhibited TCR-induced- Ca2+ mobilization and IL-2 promoter activation but not phorbol 12-myristate 13-acetate/ionomycin-induced IL-2 promoter activation, indicating its point of inhibition at the membrane proximal site of TCR signaling. Furthermore, RosA inhibited TCR-induced splenocyte proliferation as well as one-way MLR at an IC50 of 25-50 μM and inhibited cytokine expression such as IL-2 and IFN-γ. Here, we first report RosA as an inhibitor of TCR-signaling and subsequent T cell proliferation.