"Myotonic dystrophy type 1 (DM1), one of the most frequent muscular diseases in adults, is a multisystemic disorder characterized by an extremely variable clinical course. The disease manifestations include myotonia, progressive muscle weakness and wasting, as well as cardiac and cognitive impairments. DM1 is caused by a CTG trinucleotide repeat expansion in the 3’UTR of the DMPK gene. Mutated DMPK transcripts harboring expanded repeats (CUGexp-RNA) aggregate into nuclear foci that sequester RNA-binding proteins of the Muscleblind-like (MBNL) family leading to their loss of function, RNA processing misregulation and ultimately, to clinical symptoms. This mechanism plays a central role in DM1 pathogenesis. However, consequences of the depletion or functional loss of MBNL proteins in adult and fully mature skeletal muscles, independently from developmental changes intrinsic to MBNL knockout mouse models, were not examined so far. Here we designed an AAV strategy to silence both MBNL1 and MBNL2 by RNAi, specifically in skeletal muscle fibers of adult wild-type mice. We showed that the depletion of MBNL proteins leads to progressive histological and molecular skeletal muscle abnormalities reminiscent of DM1 patients’ biopsies, demonstrating that MBNL proteins are required for skeletal muscle fiber homeostasis. In addition, vital labelling of replicating DNA revealed satellite cell activation associated with nuclei internalization in MBNL-depleted muscle fibers, suggestive of an active regenerative process or of a compensatory regulation of muscle mass. Currently, transcriptomic analyses using RNA sequencing are performed at different timepoints to identify RNA processing and signaling pathways initially impaired and involved in the DM1 skeletal muscle phenotype. In conclusion, our AAV-based mouse model provides a suitable platform to study the progressive impairment of an adult skeletal muscle following functional loss of MBNL proteins triggered by the expression of CUGexp-RNA in muscle fibers. On-going molecular analyses aim at identifying new DM1 specific targets and biomarkers of the disease.