Blebbistatin is a recently discovered small molecule inhibitor of myosin II, showing high affinity and selectivity. This study investigates its mechanism of inhibition. Blebbistatin does not compete with nucleotide binding but preferentially binds to the ATPase intermediate with ADP and phosphate bound, slowing phosphate release. It blocks myosin heads in a products complex with low actin affinity, preventing rigid actomyosin cross-linking. Molecular simulations identify the aqueous cavity between the nucleotide pocket and the actin-binding interface as the binding site. Blebbistatin's ability to block myosin II in an actin-detached state makes it useful in muscle physiology and structural studies. The inhibitor's selectivity and high affinity for several class II myosins suggest its potential as a tool in cell motility and muscle physiology research.Blebbistatin is a recently discovered small molecule inhibitor of myosin II, showing high affinity and selectivity. This study investigates its mechanism of inhibition. Blebbistatin does not compete with nucleotide binding but preferentially binds to the ATPase intermediate with ADP and phosphate bound, slowing phosphate release. It blocks myosin heads in a products complex with low actin affinity, preventing rigid actomyosin cross-linking. Molecular simulations identify the aqueous cavity between the nucleotide pocket and the actin-binding interface as the binding site. Blebbistatin's ability to block myosin II in an actin-detached state makes it useful in muscle physiology and structural studies. The inhibitor's selectivity and high affinity for several class II myosins suggest its potential as a tool in cell motility and muscle physiology research.