Nuclear positioning is a determining event in several cellular processes, such as fertilization, cell migration, and cell differentiation. The structure and function of muscle cells, which contain hundreds of nuclei, have been shown to rely in part on proper nuclear positioning. Remarkably, in the course of muscle differentiation, nuclear movements along the myotube axis might represent the event required for the even positioning of nuclei in the mature myofiber. Here we analyze nuclear behavior, time in motion, speed, and alignment during myotube differentiation and temporal interference of cytoskeletal microtubule-related motors. Using specific inhibitors, we find that nuclear movement and alignment are microtubule dependent, with 19 microtubule motor proteins implicated in at least one nuclear behavior. We further focus on Kif1c, Kif5b, kif9, kif21b, and Kif1a, which affect nuclear alignment. These results emphasize the different roles of molecular motors in particular mechanisms.