Tissue inhibitor of metalloproteinase-1 (TIMP-1) plays various roles in cell growth in different cell types. However, few studies have focused on TIMP-1's effect on fibroblast cells. In this study, we investigated the effects of TIMP-1 overexpression on NIH3T3 fibroblast proliferation and potential transduction signaling pathways involved. Overexpression of TIMP-1, by transfection of the pLenti6/V5-DESTTIMP-1 plasmid, significantly promoted NIH3T3 proliferation as determined by the BrdU array. Neither 5 nor 15 nM GM6001 (matrix metalloproteinase system inhibitor) affected NIH3T3 proliferation, but 45 nM GM6001 inhibited proliferation. TIMP-1 overexpression activated the p-Akt pathway, but not the p-ERK or p-p38 pathway. In TIMP-1-transfected cells, cyclinD1 was upregulated and p21CIP1 and p27(KIP1) were downregulated, which promoted cell entry into the S and G2/M phases. The PI3-K inhibitor LY294002 abolished the TIMP-1-induced effects. Overexpression of intracellular TIMP-1 stimulated NIH3T3 fibroblast proliferation in a matrix metalloproteinase (MMP)-independent manner by activating the p-Akt pathway and related cell cycle progression.