Gomisin A (GA) is an active ingredient of the fruits of Schisandra chinensis which has been widely used as a tonic in traditional Korean medicine. GA induces not only endothelium-dependent but also endothelium-independent relaxation in an isolated rat's thoracic aorta. This study was aimed to investigate the molecular mechanism by which GA induces endothelium-independent vasorelaxation. Rat aortic rings were denuded of endothelium, mounted in organ baths, and subjected to contraction or relaxation. We measured the amount of GTP RhoA as well as the phosphorylation level of 20 kDa myosin light chains (MLC₂₀), myosin phosphatase-targeting subunit 1 (MYPT1) and protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light-chain phosphatase of 17 kDa (CPI17). Pretreatment with GA dose-dependently inhibited the concentration-response curves in response to sodium fluoride (NaF) or thromboxane A(2) agonist U46619, but not to phorbol 12, 13-dibutyrate (PDBu). GA decreased the activation of RhoA as well as the phosphorylation level of MLC₂₀, MYPT1(Thr₈₅₅), and CPI17 induced by 8.0 mM NaF or 30 nM U46619. However, K+ channel blockers such as glibenclamide, apamin, or charybdotoxin did not affect the vascular relaxation induced by GA. Furthermore, GA did not affect the level of phosphorylation of CPI17 induced by PDBu. GA reduces vascular contraction through inhibition of RhoA/Rho-kinase pathway in endothelium-denuded rat aorta.