Under proper stimulation, mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into myocytes, chondrocytes, adipocytes, osteoblasts, and tenocytes, both in vivo and in vitro. Despite the promising results of myogenic differentiation induced by 5-azacytidine, the molecular mechanisms underlying myogenic differentiation from MSCs remain poorly understood. To achieve effective regeneration of injured myocardium, it is important to find a more physiological way of improving the in situ myogenic differentiation of MSCs. In this study, we investigated the effect of phorbol myristate acetate (PMA), a PKC activator, on myogenic differentiation of MSCs. Protein Kinase C (PKC) is one of the signal transduction components involved in the myogenic differentiation of embryonic stem cells. To confirm the effect of PMA, 1關M PMA-treated MSCs were grown for each two, five or nine days. In sandwich ELISA, the expression of cardiac-specific markers (cardiac troponin T, myosin light chain, myosin heavy chain, NK2 transcription factor-related, locus 5, Myocyte-specific enhancer factor 2) was elevated at nine days. Though it has been known that few 慣1A receptors exist in MSCs, 慣1-adrenergic receptor subtypes were preferentially expressed in PMA-treated MSCs. Moreover, expression of the 棺-adrenergic and muscarinic receptors was increased 2-fold in PMA-treated MSCs compared to normal MSCs. The mRNA level of Ca2+-related factor (SERCA 2a; sarcoplasmic reticulum Ca2+-ATPase, LTCC; L-type Ca2+ channel) in treated MSCs was similar to that in cardiomyocytes. In the hypertrophic response to norepinephrine, activation of ERK1/2, a hypertrophic marker, was time-dependently augmented in PMA-treated MSCs. In conclusion, PMA, a PKC activator, might induce the myogenic differentiation of MSCs. This result suggests that regulation of protein kinase could be an alternative method for differentiation of stem cells.