Key points Deficiency of dysferlin causes limb‐girdle muscular dystrophy 2B and Miyoshi myopathy with cardiac involvement that leads to dilated cardiomyopathy and heart failure. The pathogenesis and pathophysiology of dysferlin cardiomyopathy are not fully understood. We studied cardiac phenotypes of young dysferlin gene knockout mice to investigate the primary pathological and pathophysiological changes. In comparison with wild‐type controls, dysferlin‐deficient cardiomyocytes showed slower Ca2+ re‐sequestration, and dysferlin deficiency blunted the β‐adrenergic effect on relaxation and pumping function of ex vivo working hearts. Dysferlin deficiency increased phosphorylation of ventricular myosin light chain 2, suggesting a compensatory response to the impaired cardiac lusitropic function. The data suggest that delayed calcium re‐sequestration and post‐translational modification of myofilament proteins may provide potential targets to develop new treatments for dysferlin cardiomyopathy. Abstract Dysferlin is a cell membrane bound protein with a role in the repair of skeletal and cardiac muscle cells. Deficiency of dysferlin leads to limb‐girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy. In cardiac muscle, dysferlin is located at the intercalated disc and transverse tubule membranes. Loss of dysferlin causes death of cardiomyocytes, notably in ageing hearts, leading to dilated cardiomyopathy and heart failure in LGM2B patients. To understand the primary pathogenesis and pathophysiology of dysferlin cardiomyopathy, we studied cardiac phenotypes of young adult dysferlin knockout mice and found early myocardial hypertrophy with largely compensated baseline cardiac function. Cardiomyocytes isolated from dysferlin‐deficient mice showed normal shortening and re‐lengthening velocities in the absence of external load with normal peak systolic Ca2+ but slower Ca2+ re‐sequestration than wild‐type controls. The effects of isoproterenol on relaxation velocity, left ventricular systolic pressure and stroke volume were blunted in dysferlin‐deficient mouse hearts compared with that in wild‐type hearts. Young dysferlin‐deficient mouse hearts expressed normal isoforms of myofilament proteins whereas the phosphorylation of ventricular myosin light chain 2 was significantly increased, implying a molecular response to the impaired lusitropic function. These early phenotypes of diastolic cardiac dysfunction and blunted lusitropic response of cardiac muscle to β‐adrenergic stimulation indicate a novel pathogenic mechanism of dysferlin cardiomyopathy.