Matrine, extracted from the Chinese traditional medicine Sophorae flavescentis, has been demonstrated to exhibit antitumor effects on numerous types of cancer in vivo and in vitro with low toxicity. However, its antitumor mechanism in rhabdomyosarcoma (RMS) cells remains unclear. In the present study, the antitumor effects of matrine and its underlying mechanisms in RMS were investigated in vitro. The results demonstrated that matrine inhibited cell proliferation, migration and invasion, and induced apoptosis of RMS cells in a dose-dependent manner. Furthermore, the expression levels of phosphorylated mitogen-activated protein kinase (p-MEK) and phosphorylated extracellular signal-regulated kinase (p-ERK) significantly decreased in RMS cells following matrine treatment. In addition, the apoptotic effects of matrine in RMS cells were partially inhibited upon MEK1 overexpression and enhanced upon combined treatment with an ERK inhibitor (U0126). In addition, the ratio of apoptosis regulator BCL-2/BAX significantly decreased following matrine treatment. In conclusion, these findings indicate that matrine inhibits cell proliferation and induces the apoptosis of RMS cells by suppressing the ERK signaling pathway, and may be a novel effective candidate for the treatment of patients with RMS.