Background: beta-adrenergic activation is able to exacerbate cardiac hypertrophy. Myosin light chain kinase (MLCK) and its phosphorylated substrate, phospho-myosin light chain 2 (p-MLC2), play vital roles in regulating cardiac hypertrophy. However, it is not yet clear whether there is a relationship between beta-adrenergic activation and MLCK in the progression of cardiac hypertrophy. Therefore, we explored this relationship and the underlying mechanisms in this work. Methods: Cardiac hypertrophy and cardiomyocyte hypertrophy were induced by pressure overload and isoproterenol (ISO) stimulation, respectively. Echocardiography, histological analysis, immunofluorescence and qRT-PCR were used to confirm the successful establishment of the models. A beta-blocker (metoprolol) and a calpain inhibitor (calpeptin) were administered to inhibit beta-adrenergic activity in rats and calpain in cardio-myocytes, respectively. The protein expression levels of MLCK, myosin light chain 2 (MLC2), p-MLC2, myosin phosphatase 2 (MYPT2), calmodulin (CaM) and calpain were measured using western blotting. A cleavage assay was performed to assess the degradation of recombinant human MLCK by recombinant human calpain. Results: The beta-blocker alleviated cardiac hypertrophy and dysfunction, increased MLCK and MLC2 phosphorylation and decreased calpain expression in pressure overload-induced cardiac hypertrophy. Additionally, the calpain inhibitor calpeptin attenuated cardiomyocyte hypertrophy, upregulated MLCK and p-MLC2 and reduced MLCK degradation in ISO-induced cardiomyocyte hypertrophy. Recombinant human calpain degraded recombinant human MLCK in vitro in concentration- and time-dependent manners, and this degradation was inhibited by the calpain inhibitor calpeptin. Conclusion: Our study suggested that beta-adrenergic activation may promote the degradation of MLCK through calpain in pressure overload-induced cardiac hypertrophy.