Oxidative stress decreases muscle’s ability to produce force by specifically altering myosin, the main contractile protein in muscle, and is associated with muscle aging as well as various muscle diseases. The molecular mechanism responsible for myosin’s functional perturbations due to oxidation remains unclear. Purified myosin samples were treated with hydrogen peroxide to induce oxidative modifications at specific amino acid residues within the myosin catalytic domain. Myosin’s ability to hydrolyze ATP and bind actin was measured. Changes in myosin structural dynamics were monitored using electron paramagnetic resonance spectroscopy (EPR). Oxidation perturbs actomyosin functional interaction and produces a change in myosin’s structure, specifically impairing the weak-to-strong transition of myosin necessary for force production. As myosin is the molecular motor that drives muscle contraction, myosin is likely one of the main targets of site-specific amino acid oxidative modification induced by oxidative stress. These modifications of myosin cause contractile dysfunction leading to a decreased ability of muscle to produce force.