The treatment of severe forms of 21-hydroxylase deficiency (21OHD) remains unsatisfactory in many respects. As a monogenic disease caused by loss-of-function mutations, 21OHD is a potential candidate for a gene therapy (GT) approach. The first step of GT is to demonstrate positive effects of the therapeutic vector in the Cyp21-/- mouse model. Thus, we tested the adrenal tropism of an AAVrh10-CAG-GFP vector (‘GFP vector’) then attempted to correct the phenotypic and biochemical alterations in Cyp21-/- mice using an AAVrh10-CAG-humanCYP21A2-HA vector (‘CYP21 vector’). Cyp21-/- mice had decreased body mass, high progesterone (4×), impaired stress response, increased adrenal expression of genes involved in steroidogenesis or ACTH signaling. Following injection of the GFP vector, Cyp21-/- mice showed abundant GFP expression in the adrenal cortex. Intravenous injection of the therapeutic CYP21 vector allowed 21OH expression in adrenal tissue, resulting in increased body weight and near normalization of urinary progesterone for more than 15 weeks, improved response to stress and restoration of near-normal expression of (several important genes) in the adrenal cortex. The adrenal tropism of AAVrh10 and the persistent correction of phenotypic and biochemical traits in Cyp21-/- mice pave a first step on the way to GT of 21OHD in humans.