Nuclear movement and positioning within cells has become an area of great interest in the past few years due to the identification of different molecular mechanisms and functions in distinct organisms and contexts. One extreme example occurs during skeletal muscle development and regeneration. Skeletal muscles are composed of individual multinucleated myofibers with nuclei positioned at their periphery. Myofibers are formed by fusion of mononucleated myoblasts and during their development, successive nuclear movements and positioning events have been described. The position of the nuclei in myofibers is important for muscle function. Interestingly, during muscle regeneration and in some muscular diseases, nuclei are positioned in the center of the myofiber. In this review, we discuss the multiple mechanisms of nuclear positioning that occur during myofiber formation and regeneration. We also discuss the role of nuclear positioning for skeletal muscle function.