Nemaline myopathy (NM) is a genetically and clinically heterogeneous disease that is diagnosed based on the presence of nemaline rods on skeletal muscle biopsy. Despite multiple genes and known mutations, the genetic heterogeneity of NM is not predictive of disease course. This suggests that unidentified secondary mechanisms exist that impact disease severity. We hypothesize that the common pathological endpoint of nemaline rods (despite diverse genetic causes) and unexplained range of muscle weakness suggest that shared secondary processes contribute to the pathogenesis of NM. We speculate that these processes can be identified through a proteome wide interrogation of a severe mouse model of NM in combination with pathway validation and structural and functional analyses. Proteomic analysis was performed on the Neb cKO mouse model in comparison to their wild-type counterpart to identify pathophysiologically relevant biological processes that may impact disease severity or provide new treatment targets. A differential expression analysis and Ingenuity Pathway core analysis predicted perturbations in several cellular processes including mitochondrial dysfunction and changes in energetic metabolism and stress-related pathways. Subsequent structural and functional studies have demonstrated abnormal mitochondrial placement, decreased respiratory function, and extremely low ATP content in Neb cKO muscles relative to wild type. Overall, our findings support a role for mitochondrial dysfunction as a novel contributor to muscle weakness in NM.