20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P450-derived arachidonic acid metabolite that has been shown to increase smooth muscle contractions and proliferation, stimulate endothelial dysfunction and activation and promote hypertension. We examined if 20-HETE contributes to microvascular remodeling in hypertension. In Sprague-Dawley rats, administration of the 20-HETE biosynthesis inhibitor, HET0016, or the 20-HETE antagonist, 20-HEDE prevented 5α-dihydrotestosterone (DHT)-induced increases in blood pressure as well as abrogated DHT-induced increases in media-to-lumen ratio (M/L), media thickness and collagen IV deposition in renal interlobar arteries. Reserpine prevented blood pressure elevation in DHT-treated rats but did not affect microvascular remodeling (M/L, media thickness and collagen deposition); under these conditions, treatment with 20-HETE antagonist attenuated microvascular remodeling, suggesting that 20-HETE contributes to DHT-induced vascular remodeling independent of blood pressure elevation. In Cyp4a14(-/-) mice, which display androgen-driven and 20-HETE-dependent hypertension, treatment with 20-HETE antagonist abolished remodeling of renal resistance arteries measured as media thickness (24±1 vs. 15±1μm) and M/L (0.29±0.03 vs. 0.17±0.01). Moreover, in the Cyp4a12 transgenic mice in which the expression of Cyp4a12-20-HETE synthase is driven by a tetracyclin-sensitive promoter, treatment with doxycycline resulted in blood pressure elevation (140±4 vs. 92±5 mmHg) and a significant increase in remodeling of renal resistance arteries (media thickness, 23±1 vs. 16±1 μm; M/L, 0.39±0.04 vs. 0.23±0.02); these increases were abrogated by co-treatment with 20-6,15-HEDE. This study demonstrated that 20-HETE is a key regulator of microvascular remodeling in hypertension; its effect is independent of blood pressure elevation and androgen levels.