Egg yolk phosvitin is one of the most phosphorylated proteins in nature, and thus has a strong metal-binding ability. The objective of this study was to evaluate the cytotoxic and antigenotoxic activities of phosvitin in vitro. Using the 3-[4,5-dimethythiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, the cytotoxicity of phosvitin was evaluated in human cancer cell lines of various tissue origins, including the cervix (HeLa), breast (MCF-7), stomach (AGS), lung (A549 and SK-MES-1), liver (HepG2), and larynx (Hep-2). The growth of all cancer cell lines was inhibited in a dose-dependent manner by phosvitin. Among the cancer cell lines tested, MCF-7 and SK-MES-1 were the least sensitive and HeLa, AGS, and HepG2 were the most sensitive to phosvitin. The 50% inhibition of cell viability values of phosvitin were 5.38, 11.57, 4.78, 6.98, 11.82, 3.93, and 9.97 mg/mL for HeLa, MCF-7, AGS, A549, SK-MES-1, HepG2, and Hep-2, respectively. The protective effects of phosvitin against DNA damage in human leukocytes indicated that phosvitin showed protective effects against the oxidative stress-induced DNA damages in human leukocytes. These results suggested that phosvitin has a high potential to be used as an anticancer agent for humans.