Abstract: Rhabdomyosarcoma is a common soft tissue sarcoma, and has the characteristics of high malignancy, easy metastasis and difficult treatment, which seriously threatens the health. In this study, the cell scratch test was used to prove that high molecular weight hyaluronic acid could inhibit the migration of RD cells. Fluorescent quantitative RT-PCR was further used to detect the expression of different hyaluronic acid receptors. The results showed that CD44 had a high expression level in RD cells, and high molecular weight hyaluronic acid treatment could down-regulate the expression of CD44.The results of Western blot showed that high molecular weight hyaluronic acid could down-regulate the expression of CD44 and cell migration-related proteins Cdc42 and Arp2/3. At the same time, the results of immunofluorescence super-resolution microscopy showed that while the expression of CD44 was significantly down-regulated, the filamentous and branched microfilaments of the plasma membrane edge in RD cells depolymerized, indicating that high molecular weight might inhibit the migration and movement of RD cells by inhibiting the formation of pseudopodia, which could provide new ideas and strategies for the treatment of rhabdomyosarcoma.