Objective To elucidate the role and mechanisms of inhibiting Ca²⁺/calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) in cardiac hypertrophy and apoptosis in pressure overload-induced heart failure mice. Methods Twenty-four male C57BL/6 mice aged 6-8 weeks were evenly randomized into four groups:sham operation group, sham operation+ KN-93 group, transverse aortic constriction (TAC) group, and TAC+KN-93 group. The body and heart weights of mice in each group were recorded, and the heart weight index (HWI) was calculated; the geometry and function of mouse heart were evaluated by echocardiography; the cross-sectional area, fibrosis degree and apoptosis level of mouse cardiomyocytes were detected by triticum vulgaris lectin (WGA), Masson's trichrome and TUNEL staining, respectively. The mRNA expression of atrial natriuretic peptide (ANP) was determined by qRT-PCR; Western blotting was used to detect the protein expression of ANP, brain natriuretic peptide (BNP), β-myosin heavy chain (β-MHC), cleaved Caspase 3, cleaved Caspase 9, B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-2-related X protein (Bax), phosphorylated-Ca²⁺/calmodulin-dependent protein kinase Ⅱ (p-CaMKⅡ) and sirtuin3 (Sirt3). Results The HWI, left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) were higher (all P<0.05), while the left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) were lower in TAC group compared with those in the sham group (both P<0.05). The cardiomyocyte cross-sectional area, the ANP mRNA level and the protein expressions of ANP, BNP, and β-MHC, and the myocardial fibrosis and cardiac apoptosis were significantly increased in TAC group compared with the sham group (all P<0.05). TAC group also had significantly increased protein expression of apoptosis-related proteins (cleaved Caspase 3, cleaved Caspase 9 and Bax) and decreased anti-apoptosis protein Bcl-2 compared with the sham group (all P<0.05); moreover, the ratio of p-CaMKⅡ/CaMKⅡ was decreased and the expression of Sirt3 was significantly increased in TAC group (both P<0.05). However, the above indexes were significantly reversed in TAC+KN-93 group compared with those in TAC group after the administration of KN-93, a CaMKⅡ inhibitor (all P<0.05). Conclusion Inhibition of CaMKⅡ can alleviate cardiac dysfunction and inhibit cardiac hypertrophy and apoptosis in pressure overload-induced heart failure mice by regulating Sirt3.