Left ventricular hypertrophy, diastolic dysfunction and fibrosis are the main features of hypertrophic cardiomyopathy (HCM). Guidelines recommend β-adrenoceptor or Ca2+ channel antagonists as pharmacological treatment. The Ca2+ channel blocker diltiazem recently showed promising beneficial effects in pre-clinical HCM, particularly in patients carrying MYBPC3 mutations. In the present study we evaluated whether diltiazem could ameliorate or reverse the disease phenotype in cells and in vivo in an Mybpc3-targeted knock-in (KI) mouse model of HCM. Sarcomere shortening and Ca2+ transients were measured in KI and wild-type (WT) cardiomyocytes in basal conditions (1-Hz pacing) and under stress conditions (30 nM isoprenaline,5-Hz pacing) with or without pre-treatment with 1 μM diltiazem. KI cardiomyocytes exhibited lower diastolic sarcomere length (dSL) at baseline, a tendency to a stronger positive inotropic response to isoprenaline thanWT, a marked reduction of dSL and a tendency towards arrhythmias under stress conditions. Pre-treatment of cardiomyocyteswith 1μMdiltiazemreduced the drop in dSL and arrhythmia frequency in KI, and attenuated the positive inotropic effect of isoprenaline. Furthermore, diltiazem reduced the contraction amplitude at 5 Hz but did not affect diastolic Ca2+ load and Ca2+ transient amplitude. Six months of diltiazem treatment of KI mice did not reverse cardiac hypertrophy and dysfunction, activation of the fetal gene program or fibrosis.