The purpose of this study was to examine whether the nitric oxide donor S-nitrosoglutathione (GSNO) relaxes canine tracheal smooth muscle (CTSM) strips by decreasing Ca2+ sensitivity [i.e., the amount of force for a given intracellular Ca2+ concentration ([Ca2+](i))]. We further investigated whether GSNO decreases Ca2+ sensitivity by altering the relationship between regulatory myosin light chain (rMLC) phosphorylation and [Ca2+](i) and the relationship between force and rMLC phosphorylation. GSNO (100 μM) relaxed intact CTSM strips contracted with 45 mM KCl by decreasing Ca2+ sensitivity in comparison to control strips without significantly decreasing [Ca2+](i). GSNO reduced the amount of rMLC phosphorylation for a given [Ca2+](i) but did not affect the relationship between isometric force and rMLC phosphorylation. These results show that in CTSM strips contracted with KCl, GSNO decreases Ca2+ sensitivity by affecting the level of rMLC phosphorylation for a given [Ca2+](i), suggesting that myosin light chain kinase is inhibited or that smooth muscle protein phosphatases are activated by GSNO.