Hexose-6-phosphate dehydrogenase (H6PD) is a sarcoplasmic reticulum (SR) resident enzyme that metabolizes glucose-6-phosphate and generates NADPH that drives the activation of glucocorticoids by 11β-hydroxysteorid dehydrogenase type 1. H6PD KO mice exhibit improved skeletal muscle insulin sensitivity increased glucose uptake and glycogen storage. However, H6PD KO mice also develop a myopathy with switching from Type II to Type I fibers. Affected muscles have apparently normal sarcomeric structure but contain large intrafibrillar membranous vacuoles indicating defects in the SR. To better define the phenotype we analyzed RNA from tibialis anterior (TA) and soleus muscles of WT and KO mice prior to overt myopathy using affymetrix microarrays. We performed three biological replicates for each group, consisting of pools of mRNA from four mice. RNA was verified and hybridized onto mouse 430 2.0 genome arrays. Data were normalized using GEPAS-Express and a local-pooled-error analysis and False Discovery Rate (FDR) correction applied to identify significant differentially expressed genes. Using a threshold FDR of <0.05, 269 genes differed in expression between WT and KO TA. Only 56 genes differed in soleus. To identify functional relationships among differentially-expressed genes, we queried a knowledge base showing 11 minimally-overlapping sub networks of interrelated genes. The highest-scoring sub network was enriched (P=1.3 x 10−6) for genes with known functions in the SR unfolded protein response pathway. Merging of related networks yielded a network containing 65 of the 269 genes dysregulated in TA. Four genes involved in the SR stress responses were validated by Real time-PCR and western blotting (BiP, GRP94, CALR, DDIT3). All 4 genes were overexpressed >5-fold, increasing further in older mice. The myopathy may be a result of changed glucose metabolism and altering SR redox state thereby impairing protein folding. These studies define a metabolic pathway linking SR stress to skeletal muscle integrity.