The comprehension of muscle tissue formation and regeneration is essential to develop therapeutic approaches against muscle diseases or loss in muscle mass and strength during ageing or cancer. One of the critical steps in muscle formation is the fusion of muscle cells to form or regenerate muscle fibres. To identify new genes controlling myoblast fusion, we undertook an siRNA screen in c2c12 myoblasts and found that N-alpha-acetyltransferase 15 (Naa15) knockdown enhanced c2c12 myoblast fusion suggesting that Naa15 negatively regulated myogenic cell fusion. We identified two Naa15 orthologous genes in zebrafish genome: naa15a and naa15b. These two orthologs are both expressed in myogenic domain of the somite. Knockdown of zebrafish naa15a and naa15b genes induced a “U” shaped segmentation of the myotome and alteration of myotome boundaries resulting in the formation of abnormally long myofibres spanning adjacent somites. Taken together these results show that Naa15 regulates myotome formation and myogenesis in fish.