The C3H10T1/2 cells are considered mesenchymal stem cells (MSCs) because they can be induced to become the progenitor cells for myocytes, adipocytes, osteoblasts, and chondrocytes by the DNA methyltransferase inhibitor 5'-azacytidine. In this study, we determined the effect of growth hormone (GH) on the myogenic and adipogenic lineage commitment in C3H10T1/2 cells. The C3H10T1/2 cells were treated with recombinant bovine GH in the presence or absence of 5'-azacytidine for 4 days. The myogenic commitment in C3H10T1/2 cells was assessed by immunostaining them for MyoD, the marker for myoblasts, and by determining their capacity to differentiate into the multinucleated myotubes. The adipogenic commitment in C3H10T1/2 cells was assessed by determining their ability to differentiate into adipocytes. Myotubes and adipocyteswere identified by immunocytochemistry and Oil Red O staining, respectively. C3H10T1/2 cells treated with 5'-azacytidine and GH for 4 days contained a greater percentage of MyoD-positive cells than those treated with 5'-axacytidine alone (P < 0.05). The former generated more myotubes than the latter upon induced myoblast differentiation (P < 0.05). However, C3H10T1/2 cells treated with GH alone did not form any myotubes. C3H10T1/2 cells treated with 5'-azacytidine formed adipocytes upon adipocyte differentiation induction, whereas C3H10T1/2 cells treated with GH alone did not form any adipocytes. C3H10T1/2 cells treated with both 5'-azacytidine and GH formed fewer adipocytes than those treated with 5'-azacytidine alone (P < 0.05). Both GHR and IGF-I mRNA expression in C3H10T1/2 cells were increased by 5'-azacytidine (P < 0.05), but neither was affected by GH. Overall, this study showed that GH enhanced 5'-azacytidine-induced commitment in C3H10T1/2 cells to myoblasts but inhibited 5'-azacytidine-induced commitment to preadipocytes. These results support the possibility that GH stimulates skeletal muscle growth and inhibits adipose tissue growth in part by stimulating the myogenic commitment and inhibiting the adipogenic commitment, respectively, in mesenchymal stem cells.