Hypertension is a major risk factor for cardiovascular disease. Thus, prevention of hypertension and consequent organ damage is important for reducing its incidence. In the present study, we examined the involvement of tissue inhibitor of metalloproteinase-3 (Timp-3) in N(omega)-nitro-L-arginine methyl ester (L-NAME)-induced hypertension and accompanying vascular remodeling in mice. L-NAME was orally administered to wild-type (WT) and Timp-3 knockout (KO) mice for 6 weeks, blood pressure was monitored, and histological changes in myocardial arteries were examined. After L-NAME administration, blood pressure was lower in Timp-3 KO mice than in WT mice. The coronary arteries of WT and Timp-3 KO mice were similar after L-NAME treatment and showed no differences compared to untreated control mice. However, cardiac microvessels differed histologically between WT and Timp-3 KO mice. Vascular walls were less thickened in Timp-3 KO than in WT mice, and fibrotic changes were significantly reduced in Timp-3 KO mice. Moreover, the L-NAME-induced production of reactive oxygen species in cardiac microvessels was lower in Timp-3 KO than in WT mice. These results indicate that Timp-3 plays an important role in L-NAME-induced hypertension and myocardial vascular remodeling. Our findings suggest that Timp-3 may be a novel therapeutic target for the treatment of hypertension and consequent organ damage.