Thymosin β4 (Tβ4), a ubiquitous peptide, regulates several cellular processes that include cell morphology, wound healing, and inflammatory response. Administration of exogenous Tβ4 is protective in diabetic nephropathy and in a unilateral ureteral obstruction model. However, the role of endogenous Tβ4 in health and disease conditions remains unclear. To elucidate the pathophysiological role of endogenous Tβ4 in hypertension, we examined angiotensin-II (Ang-II)–induced renal and cardiac damage in Tβ4 knockout (Tβ4 KO) mice. Tβ4 KO and wild-type C57BL/6 mice were infused continuously for 6 weeks with either vehicle or Ang-II (980 ng/kg per minute). At baseline, Tβ4 deficiency did not affect renal and cardiac function. Systolic blood pressure in the Ang-II group was similar in wild-type and Tβ4 KO mice (wild-type Ang-II, 179.25±10.11 mm Hg; Tβ4 KO Ang-II, 169.81±6.54 mm Hg). Despite the similar systolic blood pressure after Ang-II infusion, Tβ4-deficient mice had dramatically increased albuminuria and decreased nephrin expression in the kidney (P<0.005). In the heart of Tβ4 KO mice, Ang-II reduced ejection fraction and shortening fraction (ejection fraction: wild-type Ang-II 77.95%±1.03%; Tβ4 KO Ang-II 62.58%±3.25%; P<0.005), which was accompanied by cardiac hypertrophy and left ventricular dilatation. In addition, renal and cardiac infiltration of CD68 macrophages, intercellular adhesion molecule-1, and total collagen content were increased after Ang-II infusion in Tβ4 KO mice (P<0.005). Overall, our data indicate that endogenous Tβ4 is crucial in preventing tissue injury from Ang-II–induced hypertension. This study gives new insights into the protective role of endogenous Tβ4 in hypertensive end-organ damage.