Perforin is a pore-forming protein engaged mainly in mediating target T cell death and is employed by cytotoxic T lymphocytes (CTLs) and natural killer cells. However, whether it also plays a role in conventional CD4(+) T cell function remains unclear. Here we report that in perforin-deficient (PKO) mice, CD4(+) T cells are hyperproliferative in response to T cell receptor (TCR) stimulation. This feature of hyperproliferation is accompanied by the enhancement both in cell division and in IL-2 secretion. It seems that the perforin deficiency does not influence T cell development in thymus spleen and lymph node. In vivo, perforin deficiency results in increased antigen-specific T cell proliferation and antibody production. Furthermore, PKO mice are more susceptible to experimental autoimmune uveitis. To address the molecular mechanism, we found that after TCR stimulation, CD4(+) T cells from PKO mice display an increased intracellular calcium flux and subsequently enhance activation of transcription factor NFAT1. Our results indicate that perforin plays a negative role in regulating CD4(+) T cell activation and immune response by affecting TCR-dependent Ca(2+) signaling.