〔Abstract〕 Objective To investigate the effect and mechanism of down-regulation of volume-activated chloride channel protein ClC-3 on H9 c2 cardiomyocytes. Methods H9 c2 cardiomyocytes were transfected with siRNA to construct a cell model of down-regulated ClC-3; immunofluorescence staining was used to observe the surface area of H9 c2 cells; qRT-PCR was used to detect ANP, BNP and β-MHC of H9 c2 cells; the level of mitochondrial membrane potential was detected by JC-1 fluorescent probe; the image analysis system was used to study the regulatory volume decrease(RVD) of H9 c2 cardiomyocytes; the change of chloride current on H9 c2 cell membrane was detected by whole cell patch clamp technology. Results Compared with the control group, siRNA transfection of H9 c2 cells for 72 h could reduce the expression levels of ClC-3 mRNA and protein; and the down-regulation of ClC-3 expression was similar to isoproterenol, which could increase the surface area of H9 c2 cardiomyocytes and the iconic factors. Besides, it decreased the mitochondrial membrane potential of H9 c2 cells, reduced the capacity of volume regulation, and inhibited the activation of volume-activated chloride ion current. Conclusion The down-regulation of ClC-3 expression induces H9 c2 cardiomyocyte hypertrophy. Its mechanism may be related to its damage to mitochondrial function, reduction of cell volume regulation and inhibition of volume-activated chloride current activation.