CONTEXT: Type 2 diabetes mellitus (T2D) negatively affects muscle mass and function throughout life. Whether adult muscle stem cells contribute to the decrease in muscle health is not clear and insights into the stem cell niche are difficult to obtain. OBJECTIVE: To establish the upstream signaling pathway of microRNA (miR)-501, a marker of activated myogenic progenitor cells, and interrogate this pathway in muscle biopsies from patients with T2D. METHODS: Analysis of primary muscle cell cultures from mice and 4 normoglycemic humans and muscle biopsies from 7 patients with T2D and 7 normoglycemic controls using gene expression, information on histone methylation, peptide screening, and promoter assays. RESULTS: miR-501 shares the promoter of its host gene, isoform 2 of chloride voltage-gated channel 5 (CLCN5-2), and miR-501 expression increases during muscle cell differentiation. We identify platelet-derived growth factor (PDGF) as an upstream regulator of CLCN5-2 and miR-501 via Janus kinase/signal transducer and activator of transcription. Skeletal muscle biopsies from patients with T2D revealed upregulation of PDGF (1.62-fold, P = .002), CLCN5-2 (2.85-fold, P = .03), and miR-501 (1.73-fold, P = .02) compared with normoglycemic controls. In addition, we observed a positive correlation of PDGF and miR-501 in human skeletal muscle (r = 0.542, P = .045, n = 14). CONCLUSIONS: We conclude that paracrine signaling in the adult muscle stem cells niche is activated in T2D. Expression analysis of the PDGF-miR-501 signaling pathway could represent a powerful tool to classify patients in clinical trials that aim to improve muscle health and glucose homeostasis in patients with diabetes.