Metabolic dysfunction-associated steatotic liver disease (MASLD) represents a major global health challenge with limited therapeutic options. To identify new regulators of lipid metabolism, we developed a novel proteomic strategy combining organelle enrichment with a custom sORF database to explore the “dark proteome.” Using this approach, we discovered MNP33, a previously uncharacterized 28-amino acid microprotein. This novel protein protects against metabolic disease in mice by potently reducing body weight gain, improving glucose homeostasis, and decreasing hepatic triacylglycerol (TAG) accumulation. Mechanistically, MNP33 localizes to the inner mitochondrial membrane, interacts with adenine nucleotide translocase 2 (ANT2), and induces a bioenergetic remodeling characterized by increased proton leak, elevated basal respiration, and a paradoxically elevated membrane potential. This promotion of energy dissipation provides a direct basis for the observed reduction in TAG. Our findings establish MNP33 as a key regulator of hepatic lipid metabolism with therapeutic potential for treating MASLD.