The present study directly tested the hypothesis that the NHE3 (Na/H exchanger 3) in the proximal tubules of the kidney is required for the development of Ang II (angiotensin II)-induced hypertension using PT-Nhe3 (proximal tubule-specific NHE3 knockout) mice. Specifically, PT-Nhe3 mice were generated using the SGLT2-Cre/Nhe3 approach, whereas Ang II-induced hypertension was studied in 12 groups (n=5-12 per group) of adult male and female wild-type (WT) and PT-Nhe3 mice. Under basal conditions, systolic blood pressure, diastolic blood pressure, and mean arterial blood pressure were significantly lower in male and female PT-Nhe3 than WT mice (P<0.01). A high pressor, 1.5 mg/kg per day, intraperitoneal or a slow pressor dose of Ang II, 0.5 mg/kg per day, intraperitoneal for 2 weeks significantly increased systolic blood pressure, diastolic blood pressure, and mean arterial blood pressure in male and female WT mice (P<0.01), but the hypertensive response to Ang II was markedly attenuated in male and female PT-Nhe3 mice (P<0.01). Ang II impaired the pressure-natriuresis response in WT mice, whereas proximal tubule-specific deletion of NHE3 improved the pressure-natriuresis response in Ang II-infused PT-Nhe3 mice (P<0.01). AT receptor blocker losartan completely blocked Ang II-induced hypertension in both WT and PT-Nhe3 mice (P<0.01). However, inhibition of nitric oxide synthase with L-N-Nitroarginine methyl ester had no effect on Ang II-induced hypertension in WT or PT-Nhe3 mice (not significant). Furthermore, Ang II-induced hypertension was significantly attenuated by an orally absorbable NHE3 inhibitor AVE0657. In conclusion, NHE3 in the proximal tubules of the kidney may be a therapeutical target in hypertension induced by Ang II or with increased NHE3 expression in the proximal tubules.