Myosin IIIA is expressed in photoreceptor cells and thought to play a critical role in phototransduction processes, yet its function at a molecular basis is largely unknown. Here we clarified the kinetic mechanism of the ATPase cycle of human myosin IIIA. The steady-state ATPase activity was markedly activated ~10 fold with very low actin concentration. The rate of ADP off from actomyosin IIIA was 10 times greater than the overall cycling rate, thus not a rate-determining step. The rate constant of the ATP hydrolysis step of the actin-dissociated form was very slow but the rate was markedly accelerated by actin binding. The dissociation constant of the ATP bound form of myosin IIIA from actin is submicromolar that agrees well with the low Kactin. These results indicate that ATP hydrolysis predominantly takes place in the actin bound form for acto-myosin IIIA ATPase reaction. The obtained Kactin was much lower than the previously reported one, and we found that the autophosphorylation of myosin IIIA dramatically increased the Kactin, while the Vmax was unchanged. Our kinetic model indicates that both the actin attached hydrolysis and the Pi release steps determine the over-all cycle rate of the dephosphorylated form. While the stable steady state intermediates of acto-myosin IIIA ATPase reaction are not typical strong actin binding intermediates, the affinity of the stable intermediates for actin is much higher than conventional weak actin binding forms. The present results suggest that myosin IIIA can spend a majority of its ATP hydrolysis cycling time on actin.