〔Abstract〕 Objective To investigate the expression of Rac1 and Cdc42 in rhabdomyosarcoma(RMS) cells and their effect on proliferation, invasion, migration and apoptosis of RMS cells. Methods The expressions of Rac1 and Cdc42 were detected by qRT-PCR and Western blot in RMS cells and human skeletal muscle cells. The Rac1 and Cdc42 overexpression plasmids were constructed, and transfection efficiency was detected by qRT-PCR and Western blot. The effects of Rac1 and Cdc42 on the proliferation, invasion, migration and apoptosis of RMS cells were detected by CCK-8 assay, transwell system, wound-healing assay and flow cytometry assay. Results The expression levels of Rac1 and Cdc42 in RMS cells were higher than those in normal skeletal muscles cells. RMS cells transfected with Rac1 or Cdc42 overexpression plasmid had higher intracellular Rac1 and Cdc42 levels than those transfected with the empty vector(EV). Compared with the control group, overexpressing Rac1 and Cdc42 in RMS cells remarkably promoted proliferation, invasion, migration and anti-apoptosis. Conclusion Rac1 and Cdc42 are highly expressed in RMS cells and promote the malignant biological behavior of RMS cells by affecting the proliferation, invasion, migration and anti-apoptotic ability of RMS cells.