Objective To explore the preventive effect of chloride channel protein 3 (ClC-3) gene overexpression on isoprenaline (ISO)-induced myocardial hypertrophy in mice and its mechanisms. Methods ClC-3 overexpression mouse model and primary cardiomyocyte model were constructed using adeno-associated virus 9 (AAV9) infection method. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of ClC-3 in mouse heart tissues and primary cardiomyocytes to determine whether the model was successfully established. Thirty-two male C57BL/6 mice were randomly divided into 4 groups:control group (normal saline intraperitoneally for 7 d), ISO group (ISO 7.5 mg·kg^-1·d^-1 intraperitoneally for 7 d), AAV9-bv+ ISO group (ISO 7.5 mg·kg^-1·d^-1 intraperitoneally for 7 d after infected AAV9 with carrying blank vector for 4 weeks) and AAV9-CLC-3+ISO group (ISO 7.5 mg·kg^-1·d^-1 intraperitoneally for 7 d after infected AAV9 with carrying ClC-3 gene for 4 weeks), with 8 mice in each group. The cardiac function of the mice was detected by echocardiography, and the heart-body weight index was calculated. The mRNA expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in myocardial tissues was detected by qRT-PCR. The morphological changes of left ventricle were observed by hematoxylineosin staining, and the changes of cardiac collagen fibers were observed by picric-sirius red (PSR) staining. Suckling mice were selected and the primary cardiomyocytes were isolated in vitro and divided into 4 groups:control group (without any intervention), ISO group (0.1 μmol/L ISO intervention for 48 h), AAV9-ClC-3 group (AAV9 carrying ClC-3 gene infection for 48 h) and AAV9-ClC-3+ISO group (AAV9 carrying ClC-3 gene infection and 0.1 μmol/L ISO intervention for 48 h). The volume activating chloride current (I_Cl,vol) of primary mouse cardiomyocytes was measured by patch clamp technique. Results Western blotting and qRT-PCR results showed that the mouse model and primary cardiomyocyte model with ClC-3 overexpression were successfully established. AAV9-mediated ClC-3 overexpression could reduce the ISO-induced increases in heart-body weight index, end-systolic interventricular septum (IVSs), systolic left ventricular posterior wall (LVPWs), diastolic left ventricular posterior wall (LVPWd), and mRNA expression of ANP and BNP in cardiac tissues, alleviate the abnormal cardiac histomorphology and cardiac fibrosis, as well as inhibit the ISO-induced decrease of I_Cl,vol in vitro. Conclusion ClC-3 overexpression can prevent ISO-induced myocardial hypertrophy in mice, which may be related to the activation of I _Cl,vol.