Mitophagy is essential for mitochondrial quality control, selectively removing damaged or superfluous mitochondria to maintain cellular health and metabolic homeostasis. While positive regulators of mitophagy are relatively well characterized, the mechanisms governing its downregulation remain less understood. In this study, we investigate the role of Saccharomyces cerevisiae Slm35 - a protein previously involved in oxidative stress response - in the regulation of mitophagy. We discovered that Slm35 is a soluble mitochondrial matrix protein and functions as a novel negative regulator of mitophagy and the mitochondrial retrograde (RTG) signaling pathway. Our results show that Slm35 modulates mitophagy through the RTG pathway, independently of Atg32 proteolytic processing by Yme1 or mitochondrial membrane potential dissipation. Notably, Slm35 is crucial for the dynamic regulation of the RTG pathway in mitophagy-inducing conditions. These findings highlight the importance of Slm35 in fine-tuning mitochondrial quality control in response to metabolic cues and suggest a critical role for dynamic RTG pathway regulation in mitophagy control.