Mutations in β-cardiac myosin, the predominant motor protein for human heart contraction, can alter power output and cause cardiomyopathy. However, measurements of the intrinsic force, velocity, and ATPase activity of myosin have not provided a consistent mechanism to link mutations to muscle pathology. An alternative model posits that mutations in myosin affect the stability of a sequestered, super relaxed state (SRX) of the protein with very slow ATP hydrolysis and thereby change the number of myosin heads accessible to actin. Here we show that purified human β-cardiac myosin exists partly in an SRX and may in part correspond to a folded-back conformation of myosin heads observed in muscle fibers around the thick filament backbone. Mutations that cause hypertrophic cardiomyopathy destabilize this state, while the small molecule mavacamten promotes it. These findings provide a biochemical and structural link between the genetics and physiology of cardiomyopathy with implications for therapeutic strategies.