Myositis is a rare acquired autoimmune disorder which results in patients experiencing profound muscle weakness, dysfunction and disability. Myositis is characterised by inflammatory cell infiltration (CD4+/CD8+ T-cells) into muscle and the presence of an array of circulating myositis-specific and -associated autoantibodies (1). A key diagnostic feature of myositis is the overexpression of major histocompatibility complex (MHC) I upon the surface of muscle fibres. MHC I overexpression persists in the absence of inflammatory cells or overt disease, where patients remain profoundly weak (2). Moreover, genome wide single nucleotide polymorphism association studies have identified the MHC region of chromosome 6 to have the strongest disease association (3). Thus, MHC has been identified as an important pathogenic factor in myositis; however, the mechanisms associated with this are poorly understood. In this study, we examine the impact of overexpressing the MHC I isoform (HLA-A2/K^b) on mitochondrial function and dynamics, as described by mitochondrial structure, in human skeletal muscle cells. Human skeletal muscle myoblasts were transfected with a mammalian HLA-A2/K^b overexpression vector using transit-X2 transfection reagent for 18 hours; transfection efficiency was determined by immunofluorescent staining with an anti-HLA class I antibody. Mitochondrial function was assessed using seahorse extracellular flux analysis. Mitochondrial structure and morphology were observed with the MitoTracker Red probe using fluorescent microscopy and quantified using a NIH ImageJ-based macro (4). Analysis for statistical significance was determined by ANOVA with Tukey’s post hoc test, data are expressed as mean ± SEM, n=4. Overexpression of MHC I results in a decline in maximal (Ctrl, 368.4 ± 43.28 vs. MHC I, 209.1 ± 39.64 pmol/min/mg; p<0.05), basal (Ctrl, 208.6 ± 39.77 vs. MHC I, 108.6 ± 38.64 pmol/min/mg), and ATP-linked respiration (Ctrl, 93.81 ± 35.7 vs. MHC I, 47.36 ± 35.57 pmol/min/mg), alongside a decline in overall reserve capacity of cells (Ctrl, 231.4 ± 27.03 vs. MHC I, 128.9 ± 58.14 pmol/min/mg). Morphological analysis and quantitation of mitochondria show significantly increased mitochondrial perimeter (p<0.05) and decreased mitochondrial solidity (p<0.05) in MHC I overexpressing cells, two predictive features of mitochondrial fission (5). Even though elongated mitochondria are present following MHC I overexpression (p<0.05), no significant changes are found in mitochondrial interconnectivity, a vital characteristic of mitochondrial fusion events (4). These findings suggest that overexpression of MHC I induces mitochondrial dysfunction by altering mitochondrial bioenergetics and biogenesis. Overall, this study provides an insight into the mechanisms by which MHC I may induce dysfunction in myositic muscle.