Protein quality control is essential for maintaining cellular proteostasis, primarily orchestrated through the ubiquitin–proteasome system (UPS) and the autophagy–lysosomal pathway. While deubiquitinating enzymes (DUBs) are well-known regulators of the UPS, their roles in autophagy remain less understood. In this study, we identified two DUBs, Leon/USP5 and dUsp45/USP45, as critical modulators of autophagy and lysosomal function in Drosophila and mammalian cells. Knockdown of Leon/USP5 significantly increased autophagosome formation and autophagic flux under nutrient-rich conditions, and genetic analysis revealed that Leon suppresses Atg1-induced cell death. Furthermore, Leon/USP5 physically interacts with the autophagy-initiating kinase Atg1/ULK1. Depletion of Leon/USP5 led to increased protein levels of Atg1/ULK1, indicating Leon/USP5 as a novel inhibitory regulator of autophagy. Separately, we identified a novel role for dUsp45/USP45 in autophagy. Knockdown of dUsp45 enhanced autophagic flux, whereas its overexpression impaired autophagy, caused the formation of enlarged autophagosomal structures. Mechanistically, USP45 disrupts lysosomal acidification and the localization of V-ATPases to lysosomes by altering actin organization. Taken together, our findings reveal distinct mechanisms by which USP5 and USP45 fine-tune autophagic activity, highlighting the broader role of DUBs in maintaining cellular homeostasis.