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Today septins are considered as the fourth component of the cytoskeleton with the Septin-7 isoform playing a critical role in the formation of higher order structures. While its importance has already been confirmed in several intracellular processes of different organs, very little is known about its role in skeletal muscle. Here, using Septin-7 conditional knock-down mouse model, the C2C12 cell line, and enzymatically isolated adult muscle fibers the organization and localization of septin filaments is revealed, and an ontogenesis-dependent expression of Septin-7 is demonstrated. KD mice displayed a characteristic hunchback phenotype with skeletal deformities, reduction in vivo and in vitro force generation, and disorganized mitochondrial networks. Furthermore, knock-out of Septin-7 in C2C12 cells resulted in complete loss of cell division while KD cells provided evidence that Septin-7 is essential in proper myotube differentiation. These and the transient increase in Septin-7 expression following muscle injury demonstrate its vital contribution to muscle regeneration and development.