Myositis is a group of rare autoimmune diseases with progressive skeletal muscle weakness and dysfunction. Morphological features of the disease include inflammatory infiltration (CD4+/CD8+ T-cells), the presence of circulating autoantibodies, and interferons (IFN) upregulation in muscle cells (1). Specifically, patients with dermatomyositis (DM) have shown strong Type I IFN-alpha and -beta signature (2). Overexpression of major histocompatibility complex (MHC) I in muscle fibres is a key pathological feature, which is observed in the absence of inflammation or overt disease, while muscle weakness persists (3); this has been attributed to other non-immune mediated mechanisms, including mitochondrial abnormalities (4). Even though both IFN signature and MHC I have been identified to play an important role in DM, their downstream combinational effects remain to be revealed. The aim of this study was to examine the impact of MHC I overexpression in presence or absence of Type I IFNs, on mitochondrial function. Human skeletal muscle myoblasts were transfected with a mammalian HLA-A2/K^b overexpression vector, a MHC I isoform, and treated with Type I IFNs (100 ng/ml) for 18 hours. Mitochondrial and function, membrane potential and superoxide generation were assessed using seahorse extracellular flux analysis (n=4), JC-1 (n=6), and MitoSOX Red (n=8), respectively. Statistical analysis was performed by ANOVA with Dunnett’s post-hoc test. MHC I overexpression results in decreased mitochondrial respiration, including maximal, basal, and ATP-linked respiration, alongside with a decline in overall reserve capacity of cells; which was significantly compounded in the presence of Type I IFNs. Specifically, MHC I overexpressed myoblasts treated with IFN-beta show significant decrease in maximal respiration (p=0.0223), ATP production (p=0.02), and proton leak (p=0.01). Myoblasts overexpressing MHC I and treated with IFN-alpha show significant reduction in maximal respiration (p=0.0403) and proton leak (p=0.01). The reduction in proton leak is further supported by mitochondrial membrane hyperpolarisation, which is significantly observed in presence of Type I IFNs (IFN-alpha, p=0.007; IFN-beta, p<0.001). Moreover, cells overexpressing MHC I induce significant increase in mitochondrial superoxide generation (p=0.0448). Data suggest that MHC I and Type I IFNs have a strong combinational effect on mitochondrial function, providing further insights into DM pathogenesis.