Lactoferrin (LF) is an iron binding glycoprotein of the transferrin family with a wide spectrum of biological effects, including anti-cancer activity. However, the detailed molecular mechanisms of anti-cancer activity of LF have not been fully determined. In this study, we tried to clarify cytotoxic functions of LF on various cell lines under hypoxic conditions and elucidate those molecular mechanisms. Cytotoxic activity of LF on cell lines was found to have a range of sensitivities. Hypoxia decreased sensitivity to LF in KD (lip fibroblast) but increased that in HSC2 (oral squamous cell carcinoma). Expression analyses further revealed that LF treatments increased hypoxic HIF-1α, -2α and p53 proteins in KD but attenuated them in HSC2 cells, and decreased HIF-1 target gene, , in KD but increased it in HSC2, suggesting a possible relationship between LF-modified expression and HIF-α protein. MTT assay strikingly demonstrated that cells expressing mutant-type p53 (MT5) were more sensitive to LF than control HepG2 (hepatoma), suggesting an important role of the p53 signal. Knock-down of (p53 gene) interestingly reduced sensitivity to LF in HepG2, suggesting that p53 may be a target of LF cytotoxic activity. Further analyses with a ferroptosis promoter or inhibitor demonstrated that LF increased in hypoxic MT5, suggesting LF-induced ferroptosis in cells expressing mutant-type p53. In conclusion, hypoxia was found to regulate cytotoxic activities of LF differently among various cell lines, possibly through the p53 signaling pathway. LF further appeared to regulate ferroptosis through a modification of expression.