Nitric oxide (NO) deficiency induced by the NO synthase (NOS) inhibitor N^G-nitro-l-arginine-methyl ester (l-NAME) resulted in hypertension. l-citrulline (CIT) can be converted to l-arginine to generate NO. We examined whether maternal CIT supplementation can prevent l-NAME-induced programmed hypertension. Pregnant Sprague-Dawley rats were assigned to four groups: control, l-NAME, control citrulline (CIT), and l-NAME citrulline (l-NAME CIT). Pregnant rats received l-NAME administration at 60 mg/kg/day subcutaneously during pregnancy alone or with additional 0.25% l-citrulline solution in drinking water during the whole period of pregnancy and lactation. Male offspring were sacrificed at 12 wk of age. l-NAME exposure during pregnancy induces hypertension in the 12-wk-old offspring. Maternal CIT therapy prevented l-NAME-induced programmed hypertension, which was associated with a decreased asymmetric dimethylarginine (ADMA) concentration and an increased l-arginine-to-ADMA ratio in the kidney, increased urinary cGMP levels, and decreased renal protein levels of type 3 sodium hydrogen exchanger (NHE3). Together, our data suggest that the beneficial effects of CIT supplementation are attributed to its ability to increase NO level in the kidney and inhibition of NHE3 expression. Our results suggest that supplementing CIT in pregnant women with NO deficiency can improve fetal development and prevent programmed hypertension.