Nitric oxide (NO) is essential for optimal myofilament function of the rat diaphragm in vitro during active shortening. Little is known about the role of NO in muscle contraction under hypoxic conditions. Hypoxia might increase the NO synthase (NOS) activity within the rat diaphragm. We hypothesized that NO plays a protective role in isotonic contractile and fatigue properties during hypoxia in vitro. The effects of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA), the NO scavenger hemoglobin, and the NO donor spermine NONOate on shortening velocity, power generation, and isotonic fatigability during hypoxia were evaluated (Po2 ∼ 7 kPa). L-NMMA and hemoglobin slowed the shortening velocity, depressed power generation, and increased isotonic fatigability during hypoxia. The effects of L-NMMA were prevented by coadministration with the NOS substrate L-arginine. Spermine NONOate did not alter isotonic contractile and fatigue properties during hypoxia. These results indicate that endogenous NO is needed for optimal muscle contraction of the rat diaphragm in vitro during hypoxia.