Diabetes mellitus complicates pregnancies, leading to diseases in adult life in the offspring. Asymmetric dimethylarginine (ADMA) is increased in diabetes mellitus, kidney disease, and hypertension. We tested whether maternal diabetes causes increased ADMA in rats, resulting in kidney disease and hypertension in the adult offspring, and whether these can be prevented by maternal citrulline supplementation. Newborn female and pregnant Sprague-Dawley rats were injected with streptozotocin (STZ), which made up the nSTZ and STZ models, respectively. For the STZ model, 4 groups of male offspring were killed at age 3 months: the control, STZ, and Cit and STZ+Cit (control and STZ rats treated with 0.25% l-citrulline solution, respectively) groups. The nSTZ rats had lower nephron numbers. The renal level of ADMA was higher in the nSTZ rats than in controls. The STZ group developed kidney injury, renal hypertrophy, and elevated blood pressure at the age of 12 weeks. These conditions were found to be associated with increased ADMA levels, decreased nitric oxide (NO) production, and decreased dimethylarginine dimethylaminohydrolase (DDAH) activity in the kidney. In addition, ADMA caused a nephron deficit in cultured rat metanephroi. Maternal citrulline supplementation prevented hypertension and kidney injury, increased the renal DDAH-2 protein level, and restored the levels of ADMA and NO in the STZ+Cit group. Reduced nephron number and increased ADMA contribute to adult kidney disease and hypertension in offspring of mothers with STZ-induced diabetes. Manipulation of the ADMA-NO pathway by citrulline supplementation may be a potential approach to prevent these conditions.