Abstract: Tumor necrosis factor α (TNFα) has multiple biological functions; however, its regulatory roles in myogenesis are not fully understood. In this study, we aimed to clarify its functions in myoblasts proliferation, differentiation, migration, and myotube fusion in primary myoblasts or C2C12 cells. To this end, we constructed TNFα muscle-conditional knockout (TNFα-CKO) mice and thereafter, compared them with flox mice to clarify the effect of TNFα knockout on skeletal muscles. The results obtained showed phenotypes, including faster muscle development, stronger regenerative capacity, and higher exercise endurance for TNFα-CKO mice than for flox mice, but no differences in major visceral organs and skeletons were observed. And using label-free proteomic data, we noted that TNFα-CKO altered the distribution of some muscle development-related proteins, such as Hira, Casz1, Casp7, Arhgap10, Gas1, Diaph1, Map3k20, Cfl2, and Igf2 in the nucleus and cytoplasm. Additionally, GSEA analysis showed that TNFα-deficiency induced positive enrichment in oxidative phosphorylation and MyoD targets and negative enrichment in JAK-STAT signaling. These findings suggest that TNFα-CKO positively regulates muscle growth and development, possibly via these abovementioned proteins and pathways.