PPP2R3A plays a key role in the cardiac pathological and physiological processes, yet little is known about how the protein involved in normal myocardium formation and the protein-protein interaction pathways involved. To address this issue, we investigated the role of PPP2R3A in cardiac myocytes and identify PPP2R3A specific protein interaction partners to accelerate the developmental process of the mechanistic studies.PPP2R3A-silenced primary myocardial cell of neonatal rats and H9c2 cells were established by infecting shRNA lentiviral particles. RT-PCR and Western blot were used to determine the expression of PPP2R3A and silencing efficiency. The cell viability was analyzed by CCK-8 kit, then the cell cycles and apoptosis assays were detected by flow cytometry. Novel protein-protein interactions of PPP2R3A were detected by Yeast Two-Hybrid assays using a high-quality human primary cardiomyocyte cDNA library.PPP2R3A-silencing rat primary cardiomyocytes and H9c2 cells were established successfully, and the expression of PPP2R3A was downregulated significantly as confirmed by RT-PCR and Western blot. PPP2R3A silencing can inhibit the myocardial cell proliferation, arrest the cell cycle in the S phase and promote the cardiomyocytes apoptosis. 19 potential candidates like COL1A2, GIPC1and BCL6 specifically interact with PPP2R3A, and COL1A2 was the highest screening frequency, covering 12.5% of the 24 clones. These partners are highly enriched in signaling pathways associated with a variety of cellular processes.A series of studies have discovered that PPP2R3A was closely associated with heart failure and arrhythmia. Our data further confirmed PPP2R3A plays an important role in the cardiomyocytes and PPP2R3A in the regulation of cardiac events via its interaction partners. Therefore, it is a potential therapeutic target for the disease.