The perilipin (PLIN) family of proteins reside on, or near, intracellular lipid droplets and play a major role in the regulation of lipid metabolism in most tissues. PLIN5 is highly expressed in tissues that have a high capacity for fatty acid metabolism, especially skeletal muscle. The aim of this study was to delineate the role of PLIN5 on substrate metabolism in muscle. Satellite cells were isolated from the major hind-limb muscles of wild type (Plin+/+) and whole body Plin5 null (Plin-/-) mice using an explant culture method. Cells were differentiated into primary myotubes and lipid and glucose metabolism were assessed using radiometric techniques. PLIN5 deletion did not affect the oxidation or storage of extracellular-derived fatty acids. In contrast, PLIN5 deletion increased the lipolysis of intramyocellular triacylglycerol and the oxidation of the liberated fatty acids. Increasing intracellular fatty acid flux can modulate metabolic phenotypes, prompting us to investigate the effect of PLIN5 on mitochondrial respiration using the Seahorse XFe24 Flux Analyser. Mitochondrial oxygen consumption rate was not different under basal or maximally stimulated conditions in Plin5-/- vs. Plin+/+ myotubes, suggesting that the increased fatty acid flux in myotubes is insufficient to induce transcriptional programs that enhance mitochondrial function. Changes in lipid metabolism are often accompanied by altered glucose metabolism, however, carbohydrate metabolism and glycogen synthesis were not different in Plin5-/- vs. Plin+/+ myotubes.The conclusion from these in vitro studies is that PLIN5 suppresses skeletal muscle lipolysis but that this is insufficient to impact whole-cell fatty acid and glucose oxidation. Ongoing studies using muscle-specific Plin5-/- mice are assessing these relationships in vivo.