Palmitoylation is the only fully reversible post-translational lipid modification that impacts 10-20% of the human proteome, but its role during spermatogenesis remains enigmatic. In this study, through generating HA-tagged Abhd10 knock-in mice, Abhd10-null mice, and combining super-resolution fluorescence imaging and electron microscopy, we identified that the S-depalmitoylase ABHD10 is a mitochondrial matrix protein, specifically expressed in testis and is essential for male fertility. Abhd10 knockout mice manifest severe sperm motility defects accompanied by malformed mitochondrial sheaths of sperm. Strikingly, mitochondrial proteomic revealed that ABHD10 deficiency primarily disrupts respiratory chain complexes. Further, mass spectrometry-based mitochondrial acyl - biotin exchange (ABE) assays identified that ABHD10 catalyzes S-depalmitoylation of proteins involved in aerobic respiration and respiratory electron transport, particularly PDHX—key regulators of oxidative phosphorylation. In addition, ABHD10 could form complex with PDHX, thereby catalyzing its S-depalmitoylation. Overall, this work identifies S-depalmitoylase as a key determinant of male fertility and unveils the facilitating role of ABHD10-mediated S-depalmitoylation in oxidative phosphorylation.