- Type-2 diabetes (T2D) has become a pandemic. The main impairment in T2D is skeletal muscle insulin resistance (IR) and is often associated with obesity. Despite decades of study, the mechanisms of IR have not been fully resolved. Evidence has implicated mitochondrial dysfunction as a potential cause and/or consequence of IR. A potential contributing factor is a lower ability for mitochondria to oxidize lipid substrates, particularly in the presence of other competing substrates. C2C12 mouse myoblasts are a common model used to study IR and T2D in muscle, but their intrinsic respiration patterns are unknown. We investigated the cellular respiration and mitochondrial metabolism of the C2C12 myoblast cell line in the presence of lipid and non-lipid substrates. With the use of high-resolution respirometry, we learned C2C12 myoblasts exhibit a reduced capacity to oxidize lipids compared to other substrates, but no major mitochondrial defects. These data indicate the capacity for mitochondria to oxidize lipids is lower than the total respiratory capacity of C2C12 cells, suggesting that limitations in skeletal lipid oxidation may be upstream of the electron transport system. Such characterization of C2C12 myoblasts can help identify potential regulatory factors for mitochondrial fuel metabolism in future studies using this cell line in the study of IR, T2D and mitochondrial dysfunction.