We used transgenic (TG) mice overexpressing mutant α-tropomyosin [α-Tm(Asp175Asn)], linked to familial hypertrophic cardiomyopathy (FHC), to test the hypothesis that this mutation impairs cardiac function by altering the sensitivity of myofilaments to Ca2+. Left ventricular (LV) pressure was measured in anesthetized nontransgenic (NTG) and TG mice. In control conditions, LV relaxation was 6,970 ± 297 mmHg/s in NTG and 5,624 ± 392 mmHg/s in TG mice ( P < 0.05). During β-adrenergic stimulation, the rate of relaxation increased to 8,411 ± 323 mmHg/s in NTG and to 6,080 ± 413 mmHg/s in TG mice ( P < 0.05). We measured the pCa-force relationship (pCa = −log [Ca2+]) in skinned fiber bundles from LV papillary muscles of NTG and TG hearts. In control conditions, the Ca2+ concentration producing 50% maximal force (pCa50) was 5.77 ± 0.02 in NTG and 5.84 ± 0.01 in TG myofilament bundles ( P < 0.05). After protein kinase A-dependent phosphorylation, the pCa50 was 5.71 ± 0.01 in NTG and 5.77 ± 0.02 in TG myofilament bundles ( P < 0.05). Our results indicate that mutant α-Tm(Asp175Asn) increases myofilament Ca2+-sensitivity, which results in decreased relaxation rate and blunted response to β-adrenergic stimulation.