Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disorder. Mutations in the gene (MYBPC3) encoding cardiac myosin binding protein C (cMyBP-C) are a frequent cause of HCM. Clinical as well as animal-model studies have reported sex-related differences in HCM disease onset and severity. In addition, it has been established that physiological stimuli such as exercise may elicit a sexually dimorphic cardiac response. However, less attention has been paid to the sex-specific differences in the cellular pathophysiologic mechanisms underlying HCM. Therefore, we studied functional properties of the heart and sarcomeres in male and female sedentary and exercise (exposed to 8 weeks voluntary wheel running) mice.Echocardiography and isometric force measurements in mechanically isolated left ventricular (LV) membrane-permeabilized cardiomyocytes were performed in Wild-type (WT) and heterozygous (HET) knock-in mice carrying a Mybpc3 point mutation (G>A transition) associated with HCM.The LV mass was significantly lower in female WT and HET mice (23% in WT and 25% in HET), compared to corresponding male mice. Isometric force measurements revealed a significant lower maximal generated tension (Fmax) in HET male (13.0 ±1.1 kN/m2), than in females (20.0 ±2.2 kN/m2). Exercise induced a higher fractional shortening in HET male mice, which is correlated with an increased Fmax in exercised HET males. In contrast, LV weight was significantly increased in exercised HET females compared to sedentary females (7% in WT and 15% in HET). Ca2+-sensitivity was increased in exercised male and females WT mice. Similarly, Ca2+-sensitivity was enhanced in HET females, however not in exercised HET mice.In conclusion, exercise training improved cardiac and myofilament performance particularly in HET male mice, indicating that physiological stimuli may elicit a sexually dimorphic cardiac response in heterozygous Mybpc3-targeted knock-in mice.