Metabolic dysfunction-associated steatohepatitis (MASH) is a lipid-driven mitochondrial disease, arising from simple steatotic livers and often associated with excess body weight. However, the key lipid changes that drive the disease progression remain poorly characterized. Here, we identified a significant depletion of sphingosine 1-phosphate (S1P), a bioactive sphingolipid, in human MASH, compared to normal and simple steatotic livers. This was attributed to reduced expression of its biosynthetic enzyme sphingosine kinase 2 (SphK2) in both human and mouse MASH. Hepatocyte-specific ablation of Sphk2 reduced hepatic S1P levels, impaired hepatocyte mitophagy and exacerbated MASH pathology in mice. Mechanistically, mitochondrial S1P bound prohibitin 2 at F212, a modification we term S1Pylation, which was essential for recruiting LC3 and inducing PINK1 to sustain protective mitophagy in hepatocytes. Targeted supplementation of mitochondrial S1P in hepatocytes effectively preserved hepatocyte mitophagy and prevented MASH progression in high-fat, high-cholesterol diet-induced obese mice. Collectively, these findings reveal mitochondrial S1P as a critical lipid suppressor of MASH.