In　this　study,　we　sought　to　determine　the　role　of　tRNA-derived　fragments　in　the　regulation　of　gene　expression　during　skeletal　muscle　cell　proliferation　and　differentiation.　We　employed　cell　culture　to　examine　the　function　of　mt-Ty　5＇　tiRNAs.　Northern　blotting,　RT-PCR　as　well　as　RNA-Seq,　were　performed　to　determine　the　effects　of　mt-Ty　5＇　tiRNA　loss　and　gain　on　gene　expression.　Standard　and　transmission　electron　microscopy　(TEM)　were　used　to　characterize　cell　and　sub-cellular　structures.　mt-Ty　5＇tiRNAs　were　found　to　be　enriched　in　mouse　skeletal　muscle,　showing　increased　levels　in　later　developmental　stages.　Gapmer-mediated　inhibition　of　tiRNAs　in　skeletal　muscle　C2C12　myoblasts　resulted　in　decreased　cell　proliferation　and　myogenic　differentiation;　consistent　with　this　observation,　RNA-Seq,　transcriptome　analyses,　and　RT-PCR　revealed　that　skeletal　muscle　cell　differentiation　and　cell　proliferation　pathways　were　also　downregulated.　Conversely,　overexpression　of　mt-Ty　5＇tiRNAs　in　C2C12　cells　led　to　a　reversal　of　these　transcriptional　trends.　These　data　reveal　that　mt-Ty　5＇tiRNAs　are　enriched　in　skeletal　muscle　and　play　an　important　role　in　myoblast　proliferation　and　differentiation.　Our　study　also　highlights　the　potential　for　the　development　of　tiRNAs　as　novel　therapeutic　targets　for　muscle-related　diseases.