Mitochondria play a central role in lipid metabolism and pathology in obesity and type 2 diabetes mellitus. Mitochondria have been shown to associate with lipid droplets (LDs) in multiple tissues but the functional role of these peridroplet mitochondria (PDM) is unknown. This work reveals that PDM have unique protein composition and cristae structure, and remain adherent to the LD in the tissue homogenate. We developed an approach to isolate PDM based on their adherence to LDs. Comparison of purified PDM to cytoplasmic mitochondria reveals that (1) PDM have increased pyruvate oxidation, electron transport, and ATP synthesis capacities. (2) PDM have reduced beta oxidation capacity and depart from LDs upon activation of brown adipose tissue thermogenesis and beta oxidation. (3) PDM support LD expansion as Perilipin 5-induced recruitment of mitochondria to LDs increases ATP-dependent triacylglyceride synthesis. (4) PDM maintain a distinct protein composition due to uniquely low fusion-fission dynamics. We conclude that PDM represent a segregated mitochondrial population with unique structure and function that supports triacylglyceride synthesis. We suggest that increased mitochondrial recruitment to LDs may be part of a generalized adaptive response in physiological conditions that require LD expansion, such as post-prandial lipid synthesis and storage. Furthermore, PDM-mediated LD expansion may play a role in muscle and liver injury from lipotoxicity in conditions of nutrient excess, such as obesity and hyperlipidemia. A better understanding of PDM and LD biology may therefore lead to new therapies for lipotoxic tissue injury and insulin resistance. / 2020-10-24T00:00:00Z