Neuroblastoma is a typical solid tumor common in childhood. The present study investigated the inhibitory effects of lactoferrin on the proliferation of IMR-32 neuroblastoma cells, including under X-ray irradiation. In controlledin vitroassays, it was found that lactoferrin inhibited cell proliferation, accompanied by cell membrane disruption. Furthermore, intracellular reactive oxygen species generation increased in IMR-32 cells treated with lactoferrin, causing membrane lipid peroxidation and the leakage of lactate dehydrogenase. The IC50values for cell proliferation were ~2.0 nM for doxorubicin, 2.7 mM for dibutyryl-cAMP and 45.9 µM for lactoferrin. X-ray irradiation at 1 Gy decreased cell proliferation to ~30%, which was not restored by lactoferrin. In the Fenton reaction system with iron chloride, lactoferrin increased hydroxyl radical (OH·) formation via H2O2, as confirmed by electron spin resonance spectra. On the whole, the findings of the present study indicate that lactoferrin, found abundantly in milk, may help prevent or treat neuroblastoma in infants with modest efficacy, and does not exert a protective effect against X-rays.