β-Adrenergic signaling regulates many physiological processes in skeletal muscles. A wealth of evidence has shown that β-agonists can increase skeletal muscle mass in vertebrates. Nevertheless, to date, the specific role of β-adrenergic receptors in different cell phenotypes (myoblasts, fibroblasts, and myotubes) and during the different steps of embryonic skeletal muscle differentiation has not been studied. Therefore, here we address this question through the analysis of embryonic chick primary cultures of skeletal muscle cells during the formation of multinucleated myotubes. We used isoproterenol (ISO), a β-adrenergic receptor agonist, to activate the β-adrenergic signaling and quantified several aspects of muscle differentiation. ISO induced an increase in myoblast proliferation, in the percentage of Pax7-positive myoblasts and in the size of skeletal muscle fibers, suggesting that ISO activates a hyperplasic and hypertrophic muscle response. Interestingly, treatment with ISO did not alter the number of fibroblast cells, suggesting that ISO effects are specific to muscle cells in the case of chick myogenic cell culture. We also show that rapamycin, an inhibitor of the mammalian target of rapamycin signaling pathway, did not prevent the effects of ISO on chick muscle fiber size. The collection of these results provides new insights into the role of β-adrenergic signaling during skeletal muscle proliferation and differentiation and specifically in the regulation of skeletal muscle hyperplasia and hypertrophy. © 2019 The Authors. Cell Biology International published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology.