The processes of muscle growth and development, including myoblast proliferation, migration, differentiation, and fusion, are modified by a variety of regulatory factors.MYL4plays an important role in atrial development, atrial cardiomyopathy, muscle-fiber size, and muscle development. The structural variation (SV) ofMYL4was found via the de novo sequencing of Ningxiang pigs, and the existence of SV was verified in the experiments. The genotype distribution of Ningxiang pigs and Large White pigs was detected, and it was found that Ningxiang pigs were mainly of theBBgenotype and that Large White pigs were mainly of theABgenotype. However, the molecular mechanisms behind theMYL4-mediated regulation of skeletal muscle development need to be deeply explored. Therefore, RT-qPCR, 3′RACE, CCK8, EdU, Western blot, immunofluorescence, flow cytometry, and bioinformation analysis were used to explore the function ofMYL4in myoblast development. The cDNA ofMYL4was successfully cloned from Ningxiang pigs, and its physicochemical properties were predicted. The expression profiles in six tissues and four stages of Ningxiang pigs and Large White pigs were found to be the highest in the lungs and 30 days after birth. The expression ofMYL4increased gradually with the extension of the myogenic differentiation time. The myoblast function test showed that the overexpression ofMYL4inhibited proliferation and promoted apoptosis and differentiation. The knockdown ofMYL4showed the opposite result. These results enhance our understanding of the molecular mechanisms of muscle development and provide a solid theoretical foundation for further exploring the role of theMYL4gene in muscle development.