Thrombospondin-4 (TSP-4) expression increases dramatically in hypertrophic and failing hearts in rodent models and in humans. The aim of this study was to address the function of TSP-4 in the heart. TSP-4-knockout (Thbs4−/−) and wild-type (WT) mice were subjected to transverse aortic constriction (TAC) to increase left ventricle load. After 2 wk, Thbs4−/− mice had a significantly higher heart weight/body weight ratio than WT mice. The additional increase in the heart weight in TAC Thbs4−/− mice was due to increased deposition of extracellular matrix (ECM). The levels of interstitial collagens were higher in the knockout mice, but the size of cardiomyocytes and apoptosis in the myocardium was unaffected by TSP-4 deficiency, suggesting that increased reactive fibrosis was the primary cause of the higher heart weight. The increased ECM deposition in Thbs4−/− mice was accompanied by changes in functional parameters of the heart and decreased vessel density. The expression of inflammatory and fibrotic genes known to be influential in myocardial remodeling changed as a result of TSP-4 deficiency in vivo and as a result of incubation of cells with recombinant TSP-4 in vitro. Thus, TSP-4 is involved in regulating the adaptive responses of the heart to pressure overload, suggesting its important role in myocardial remodeling. Our study showed a direct influence of TSP-4 on heart function and to identify the mechanism of its effects on heart remodeling.—Frolova, EG, Sopko, N., Blech, L., Popović, ZB, Li, J., Vasanji, A., Drumm, C., Krukovets, I., Jain, MK, Penn, MS, Plow, EF, Stenina, OI Thrombospondin-4 regulates fibrosis and remodeling of the myocardium in response to pressure overload.