RhoA/Rho kinase (ROCK) signaling plays a key role in the pathogenesis of experimental pulmonary hypertension (PH). Dehydroepiandrosterone (DHEA), a naturally occurring steroid hormone, effectively inhibits chronic hypoxic PH, but the responsible mechanisms are unclear. This study tested if DHEA was also effective in treating monocrotaline (MCT)-induced PH in left pneumonectomized rats, and if inhibition of RhoA/ROCK signaling was involved in DHEA's protective effect. Three weeks after MCT injection, pneumonectomized rats developed PH with severe vascular remodeling, including occlusive neointimal lesions in pulmonary arterioles. In lungs from these animals, we detected cleaved (constitutively active) ROCK I, as well as increases in activities of RhoA and ROCK, and in ROCK II protein expression. Chronic DHEA treatment (1%, by food for 3 wks) markedly inhibited the MCT-induced PH (mean pulmonary artery pressures after treatment with 0 and 1% DHEA were 33 ± 5 and 16 ± 1 mmHg, respectively) and severe pulmonary vascular remodeling in pneumonectomized rats. The MCT-induced changes in RhoA/ROCK-related protein expression were nearly normalized by DHEA. A 3-week DHEA treatment (1%) started 3 weeks after MCT injection completely inhibited the progression of PH (mean pulmonary artery pressures after treatment with 0 and 1% DHEA were 47 ± 3 and 30 ± 3 mmHg, respectively), and this treatment also resulted in 100% survival in contrast to 30% in DHEA-untreated rats. These results suggest that inhibition of RhoA/ROCK signaling, including the cleavage and constitutive activation of ROCK I, is an important component of DHEA's impressive protection against MCT-induced PH in pneumonectomized rats.