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Pleomorphic rhabdomyosarcoma (pRMS) is a rare and highly malignant mesenchymal tumor. Complete resection is the only curative treatment available, owing to the limited efficacy of chemotherapy and radiotherapy. Therefore, developing novel therapies for pRMS is important for improving clinical outcomes. Herein, a novel pRMS cell line, SMU-pRMS, was established for a detailed understanding of the biological characteristics of pRMS, thereby developing new therapies. A tissue sample from a surgically resected tumor of a 69-year-old patient was subjected to primary culture. The cell line was established and authenticated by evaluating the short tandem repeats of DNA microsatellites. Monolayer cultures of SMU-pRMS cells exhibited constant growth, spheroid formation, and invasiveness. These cells exhibited high chemosensitivity to eribulin. In addition, mice inoculated with SMU-pRMS cells developed tumors after 4 weeks. Therefore, the SMU-pRMS cell line is a useful tool for investigating pRMS development and evaluating novel therapeutic agents.