Obesity increases the risk for diabetes and cardiovascular disease. Genetic predisposition exacerbates environmental drivers of obesity such as energy-dense diets and a sedentary lifestyle. We have used divergently selected Fat (23% fat) and Lean (4% fat) lines of mice to identify the genes underlying adiposity. A stratified approach using quantitative trait loci (QTL; heritable genetic intervals segregating with adiposity in Fat x Lean F2 populations), transcriptomics, and comparative cross-species bioinformatics identified candidate obesity genes (Morton et al., 2016). A specific phospholipase A2 isoform (we name here PlaX), positioned in obesity (Fob)-1 QTL, exhibited ~5-fold elevated mRNA levels in the skeletal muscle of Fat mice compared to Lean mice. PlaX has been previously linked to the regulation of intracellular membrane vesicle trafficking and generation of lipid signalling mediators (Prunonosa Cervera et al., 2021). Overexpression of PlaX in C2C12 myotubes impaired cellular energetics and increased levels of the active form of AMP-activated protein kinase (AMPK). This led us to hypothesize that skeletal muscle PlaX overexpression may drive obesity by compromising myocyte energetics. To characterize the role of PlaX in skeletal muscle, we have overexpressed PlaX in L6 myotubes (n=3) using lentiviral transduction and performed RNA-sequencing on the Illumina NextSeq 2000 platform. As expected, Plax was most significantly overexpressed with a +6.1886-fold change (Adj.p= 5.84e-14) in overexpressed cells (differential gene expression analysis). Several genes with differential expression in PlaX overexpression cells were linked to mitochondrial respiration and metabolism. These included Pdk4, which was down regulated with -1.02327 fold-change (adj.p value 0.0012); and Mitochondrial calcium uptake gene (Micu1) which was down regulated with a -0.328-fold change (Adj.P= 1.16e^-11). It is known that Mitochondrial calcium uptake 1 (Micu1) negatively regulates thermogenesis and deletion of Micu1 in adiposity impairs thermogenesis and may lead to increased risk of obesity and metabolic dysfunction. Anti-Flag magnetic beads were used to perform Immunoprecipitant (IP) pull down to identify PlaX binding partners. As expected, PlaX was overexpressed in IP eluant >1*10^8. We performed Gene ontology on proteins detected in PlaX eluant (which were not detected in GFP and not treated controls) and found mitochondrial cellular compartments were overexpressed (Differentially expressed protein analysis, n=3, FDR-adjusted (adj.) p<0.02). Our IP and RNA-Sequencing data indicate the PlaX has a regulatory role on mitochondrial metabolism, and this may be a mechanism driving increased adiposity. In summary, our genetic strategy has identified a novel potential skeletal muscle driver of obesity that could be a tractable target for therapeutic development.