Objective: While the cardiac ubiquitin–proteasome system (UPS) is integral in regulating key cellular processes, experimentally inhibiting of proteasomal activity by chemical compound before IR injury implemented a significant reduction in cardiac cell death and infarct size. However, the search of more specific drug target in contrast to global pharmacologic proteasome inhibition is still actually. The present work explores the effect of specific UPS limitation by proteasome subunit b7 (PSMB7) gene silencing in neonatal cardiomyocyte culture and its effect at anoxia-reoxygenation (A/R) modeling: cell viability and apoptosis regulator gene expression. Methods: Cardiomyocytes were isolated from ventricles of 2 days old rats by collagenase/ pancreatine digestion and transfected by siRNA to PSMB7 gene or scrambled siRNA as control using electroporation procedure. A/R was carried out for 30/60 minutes. The number of living, necrotic and apoptotic cardiomyocytes was determined by double staining with Hoechst 33342 and propidium iodide. The level of mRNA expression was estimated by real-time PCR. Results: The silencing decreased PSMB7 expression in cells in 1.6 times (P<0.05 compared with control). In 24 hours after PSMB7 gene silencing the level of living cardiomyocytes was decreased significantly: 68.6% (P<0.005) compared to control (75.6%), with no reduction in the number of apoptotic cells. However, protective effect was observed after A/R modeling that exhibit minor increasing of live cells percentage: 68.8% vs. 66.6% (control with A/R modeling) but decrease in apoptotic cells: 3.7% (P<0.01) vs. 6.5% (control, A/R). PSMB7 knockdown in cardiomyocytes exhibited increase in Bcl-2 mRNA level, which is antiapoptotic protein and is known to protect against IR injury. Conclusion: Our data shows that PSMB7 downregulation has a negative effect for cardiomyocyte survival in normal condition, but it may be protective during anoxia-reoxygenation.