AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis and its activation during T cell receptor stimulation has recently been reported. In this study, we examined the role of AMPK in interleukin (IL)-2 production in T cells. Inhibition of AMPK by compound C, a specific inhibitor of AMPK or small interfering RNA of AMPKalpha1 suppressed IL-2 production in Jurkat T cells and peripheral blood lymphocytes stimulated with PMA plus ionomycin (PMA/Io) or with monoclonal anti-CD3 plus anti-CD28. We then showed that AMPK inhibition reduced PMA/Io-induced IL-2 mRNA expression and IL-2 promoter activation. Moreover, inhibition of AMPK suppressed transcriptional activation of NF-AT and AP-1, but not NF-kappaB, in PMA/Io-activated Jurkat cells. Finally, we found that compound C inhibited PMA/Io-induced phosphorylation of p38, JNK, and GSK-3beta but not of ERK. These results suggest that AMPK mediates IL-2 production by regulating NF-AT and AP-1activation during T cell stimulation.