Acute hypoxia dilates most systemic arteries leading to increased tissue perfusion. We previously have shown that at high stimulus conditions, porcine coronary artery was relaxed by hypoxia without a change in [Ca²⁺]_i (26). This Ca²⁺-desensitizing hypoxic relaxation (CHDR) was validated in permeabilized porcine coronary artery smooth muscle (PCASM) in which hypoxia decreased force and myosin regulatory light chain phosphorylation (p-MRLC) despite fixed [Ca²⁺] (9). Rho kinase-dependent phosphorylation of MYPT1 (p-MYPT1) is associated with decreased MRLC phosphatase activity, and increased Ca²⁺-sensitivity of both p-MRLC and force. We recently reported that p-MYPT1 dephosphorylation was a key effector in CDHR. In the current study, we tested the hypothesis that Rho kinase and not p-MYPT1 phosphatase is the regulated enzyme involved in CDHR. We used -toxin to permeabilize de-endothelialized PCASM. CDHR was attenuated in contractions attributable to myosin light chain kinase (in the presence of the Rho kinase inhibitor Y27632). In contrast, hypoxia relaxed contractions attributable to Rho kinase phosphorylation of MYPT1 and MRLC, or MRLC alone (in the presence of the MLCK inhibitor ML7). Using an in situ assay, we showed that Rho kinase activity, measured as thiophosphorylation of MYPT1 and MRLC, was nearly abolished by hypoxia. The in vitro activity of the catalytically active fragment of Rho kinase was not affected by hypoxia. Our evidence strongly implicates that hypoxia directly inhibits Rho kinase-dependent phosphorylation of MYPT1. This underlies the decreases in both p-MYPT1 and p-MRLC and thereby leads to the Ca²⁺-desensitizing hypoxic relaxation.