Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma of mesenchymal origin with two main variants, the embryonal, less aggressive, and the alveolar RMS, more metastatic. The role of the extracellular matrix (ECM) in the growth and migration of RMS, as in other cancers, is becoming increasingly important. This work aims to study the RMS after the silencing of the proteoglycan Glypican 3, overexpressed in RMS. Using classical 2D cell culture with RMS cell lines and 3D hyaluronic acid-based hydrogel, the involvement of Glypican 3 in adhesion, proliferation, matrix degradation, and consequent cell motility was demonstrated. Functional assays were performed with the antineoplastic drug doxurubicin and the WNT3a inhibitor, ipafricept. Both in 2D and in 3D model, cell motility and proliferation were significantly impaired after Glypican 3 silencing and inhibition of the proteoglycan releasing the sulfatase enzyme SULF2. When the in vivo cell-ECM interactions were mimicked in the hyaluronic acid-based hydrogel, Doxorubicin and ipraficept were particularly effective against the GPC3-silenced RMS cells. This study lay the fundation for a different therapeutic approach against pediatric RMS that aim to dysregulate the protein microenvironment not only beat the cancer cells.