〔Abstract〕 Objective To investigate the effect of inhibiting lipid synthesis on human skeletal rhabdomyosarcoma cells and its molecular mechanism. Methods The mRNA expression levels of lipid synthesis related genes sterol regulatory element binding protein 1(SREBP1) and squalene cyclooxygenase(SQLE) in human skeletal muscle cells(HSMC) and human skeletal rhabdomyosarcoma cells were detected by tumor gene expression database and verified by qRT-PCR. The concentration of methyl-β-cyclodextrin(mβCD) was determined by cell proliferation assay. The control group consisted of three human skeletal rhabdomyosarcoma cell lines(RD, SJCRH30, A673), while the experimental group comprised three human skeletal rhabdomyosarcoma cell lines treated with 1mmol/L mβCD. Plate clone formation assay, soft agar, colony formation assay, cell migration assay and cell invasion assay, and tumor formation in nude mice employed to assess changes in proliferation, migration, invasion, and tumor growth of human skeletal rhabdomyosarcoma cells between the control group and mβCD treatment group. The molecular mechanism of mβCD inhibiting the malignancy of human skeletal rhabdomyosarcoma cells was explored by lipoomics and triglyceride(TG) detection. Results Compared with HSMC, the expressions of lipid synthesis related genesSREBP1andSQLEsignificantly increased in human skeleton rhabdomyosarcoma cells(P<0.001). Compared with the control group, the proliferation, plate cloning, migration, invasion and colony formation ability of human skeleton rhabdomyosarcoma cells in the mβCD group significantly decreased(P<0.05). The growth of tumor volume and weight in nude mice was also significantly reduced(P<0.05). The lipidomics results and TG kit analysis revealed a significant reduction in TG content in the mβCD group compared to the control group(P<0.01). Conclusion mβCD may inhibit the malignant biological behaviors such as proliferation, migration and invasion of human skeleton rhabdomyosarcoma cells by reducing TG and other lipid metabolism.