Purpose: Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children and can be divided into two main subtypes: embryonal and alveolar RMS. Patients with metastatic disease continue to have very poor prognosis although aggressive therapies and recurrences are common in advanced localized disease. The oncogenic potential of the Notch pathway has been established in some cancers of the adult and in some pediatric malignancies. Experimental Design: A real-time PCR assay was used to ascertain the expression of several Notch pathway components in a wide panel of RMS and cell lines. Four γ-secretase inhibitors (GSIs) were tested for pathway inhibition and the degree of inhibition was assessed by analysis of Hes1 and Hey1 expression. The putative effects of Notch pathway inhibition were evaluated by wound-healing, matrigel/transwell invasion, cell-cycle, and apoptosis assays. Results: The Notch pathway was widely expressed and activated in RMS and underwent substantial inhibition when treated with GSIs or transfected with a dominant negative form of MAML1. RMS cells showed a significant decrease in its mobility and invasiveness when the Notch pathway was properly inhibited; conversely, its inhibition had no noticeable effect on cell cycle or apoptosis. Conclusion: Pharmacological or genetic blockage of the pathway significantly reduced invasiveness of RMS cell lines, thereby suggesting a possible role of the Notch pathway in the regulation of the metastatic process in RMS.