We have recently demonstrated that tissue inhibitor of metalloproteinase-3 (TIMP-3) decreases neonatal cardiomyocyte proliferation (Hammoud L, Xiang F, Lu X, Brunner F, Leco K, Feng Q. Cardiovasc Res 75: 359-368, 2007). The aim of the present study was to delineate a pathway through which TIMP-3 exerts its antiproliferative effect. Experiments were conducted on neonatal cardiomyocyte cultures and heart tissues isolated from wild-type (WT) and TIMP-3(-/-) mice. Deficiency in TIMP-3 decreased p27 expression and increased cardiomyocyte proliferation in cardiomyocytes and neonatal hearts. A TIMP-3/epidermal growth factor (EGF) receptor (EGFR)/c-Jun NH(2)-terminal kinase (JNK)/SP-1/p27 pathway was investigated. JNK phosphorylation and EGFR protein levels were increased in TIMP-3(-/-) cardiomyocytes and heart tissues. Treatment with recombinant TIMP-3 decreased JNK phosphorylation and EGFR expression/phosphorylation. Inhibition of JNK activity using SP-600125 decreased SP-1 phosphorylation, increased p27 expression, and decreased cardiomyocyte proliferation. Furthermore, treatment with the EGFR specific inhibitor PD-168393 or the EGF-neutralizing antibody decreased cardiomyocyte proliferation as well as phosphorylation of JNK and SP-1 in both WT and TIMP-3(-/-) cardiomyocytes. We conclude that TIMP-3 inhibits neonatal mouse cardiomyocyte proliferation by upregulating p27 expression. The effects of TIMP-3 are mediated via inhibition of EGFR expression/phosphorylation, and decreases in JNK and SP-1 signaling.