Mitophagy is a selective clearance of damaged or superfluous mitochondria via the autophagy-lysosome pathway. PINK1 (PTEN-induced kinase 1), a protein kinase, and Parkin, an E3 ligase, are two key regulators of mitophagy via a well-established positive feedback loop. On the other hand, the ubiquitin proteasome system (UPS) has been closely implicated in mitophagy. One key finding is that UPS-mediated degradation of outer mitochondrial membrane (OMM) proteins is essential for the progression of mitophagy and inhibition of proteasome completely blocks mitophagy. At present, the exact functional relationship between UPS and mitophagy remains elusive. In this study, we aim to elucidate the intricate relationships between UPS and the mitophagy machinery. Here, we show that inhibition of UPS by proteasome inhibitors prevents OMM protein degradation in cells treated with acute mitochondrial damage agents, and eventually blocks mitophagy. Mechanistically, proteasome inhibition delays PINK1 stabilization, Ub phosphorylation and Parkin activation. Second, we unexpectedly found that OPTN (optineurin) and NDP52 (nuclear dot protein 52), two fundamental mitophagy receptors, are prevented from recruiting to damaged mitochondria upon proteasome inhibition. Further studies will be focused on explaining the altered Ub linkage types upon proteasome inhibition. Taken together, our preliminary data reveal an intricate relationship between two key intracellular degradation pathways: UPS and mitophagy.