MicroRNAs are small noncoding RNAs that act as posttranscriptional regulators of gene expression. To identify microRNAs involved in T cell activation, we performed a microRNA array profiling with Jurkat cells. We found that microRNA-21 (miR-21), which is upregulated in many tumors by targeting a series of tumor suppressor genes to promote tumor growth, was significantly increased in activated Jurkat cells and primary CD4⁺ T lymphocytes compared with that in quiescent counterparts. By using a signaling network building tool, miR-21 was predicted regulates ERK and JNK signaling in activated Jurkat cells. Indeed, miR-21 promotes ERK and JNK signaling in activated T cells. Sprouty1, a direct target of miR-21 that has been shown an inhibitor of ERK and JNK, was also inhibited by forced miR-21 expression in activated T cells. Reciprocally, miR-21 levels were induced by MEK or JNK signaling response to T cell receptor (TCR) engagement. Furthermore, transfection with miR-21 mimic promotes activator protein 1 (AP-1) activity and interleukin-2 (IL-2) expression. These results provide a missing function of miR-21 in TCR-mediated signaling transduction in T lymphocytes, suggesting that miR-21 may augment T cell immune response by a positive feedback mechanism.