Updated project metadata. Background: Skeletal muscle crucially depends on motor innervation, and, when damaged, on the resident muscle stem cells (MuSCs). However, the role and function of MuSCs in the context of denervation remains poorly understood. Methods: Alterations of MuSCs and their myofiber niche after denervation were investigated in a surgery-based mouse model of unilateral sciatic nerve transection. FACS-isolated MuSCs were subjected to RNA-sequencing and mass spectrometry for the analysis of intrinsic changes after denervation and in vivo assays, such as Cardiotoxin-induced muscle injury or MuSC transplantation, were performed to assess MuSC functions after denervation. Bioinformatic and histological analyses were conducted to further examine MuSCs and their myofiber niche after denervation. Results: Muscle cross section analysis revealed a significant increase in Pax7 (p-value= 0.0441), Pax7/Ki67 (p-value= 0.0023), MyoD (p-value= 0.0016) and Myog (p-value= 0.0057) positive cells after denervation, illustrating a break of quiescence and commitment to the myogenic lineage. An Omics approach showed profound intrinsic alterations on the mRNA (2613 differentially expressed genes, p-value <0.05) and protein (1096 differentially abundant proteins, q-value <0.05) level of MuSCs 21 days after denervation. Skeletal muscle injury together with denervation surgery caused deregulated regeneration, indicated by the reduced number of proliferating MuSCs and sustained high levels of developmental myosin heavy chain (Sham: 1 % vs DEN: 40 % of all myofibers), at 21 days post-surgery. In a transplantation assay, MuSCs from a denervated host were still able to engraft and fuse to form new myofibers, irrespective of the innervation status of the recipient muscle. Analysis of myofibers revealed not only massive changes in the expression profile (10492 differentially expressed genes, p-value <0.05) after denervation, but it was also shown that secretion of Opn and Tgfb1 from denervated myofibers was increased 30-fold and 6000-fold, respectively. Bioinformatic analyses indicated strong upregulation of gene expression of the transcription factor Junb in MuSCs from denervated muscles (log2 fold change = 3.27). Of interest, Tgfb1 recombinant protein was able to induce Junb gene expression in vitro, demonstrating that myofiber-secreted ligands can induce gene expression changes in MuSCs, which might result in the phenotypes observed after denervation. Conclusion: Skeletal muscle denervation is altering myofiber secretion, causing MuSC activation and profound intrinsic changes, leading to reduced regenerative capacity. As MuSCs possess a remarkable regenerative potential, they might represent a promising target for novel treatment options for neuromuscular disorders and peripheral nerve injuries.