Objective To investigate the effect of cellular Fas-associated death domain-like interleukin-1β-converting enzyme-inhibitory protein (c-FLIP) antisense oligonucleotide (ASODN)-loaded nanoparticles (NP) on the human orbital rhabdomyosarcoma xenograft in nude mice, so as to assess the feasibility of nanoparticles as a gene vector. Methods The model of human orbital rhabdomyosarcoma xenograft was established in nude mice, and the tumors were injected with c-FLIP ASODN NP, c-FLIP ASODN or normal saline (NS). The tumor volume and histopathological changes of tumor were observed. Western blotting analysis and immunohistochemical analysis were used to examine the expression of c-FLIP in tumor tissues of each group. Apoptosis of tumor cells was detected using TUNEL method. Results The growth of human orbital rhabdomyosarcoma in nude mice was significantly inhibited in ASODN NP group compared with the other two groups. Western blotting analysis showed that c-FLIP protein expression in ASODN NP and ASODN groups was significantly decreased compared with NS group (P<0.05). Immunohistochemical study showed that c-FLIP expression was found in the endochylema, and the c-FLIP positive cells in ASODN NP group was significantly less than those in the other two groups (P<0.05). Tumor cell apoptosis was observed in both ASODN NP and ASODN groups, with more found in the former, and only a few apoptotic cells were found in the NS group. Conclusion c-FLIP ASODN NP can effectively inhibit the growth of human orbital rhabdomyosarcoma xenograft in nude mice, indicating that nanoparticles may serve as a safe and effective vector for ASODN.