Selenomethionine (SeMet) is the chemical form or major component of selenium used for cancer chemoprevention in several clinical trials. However, evidence from experimental studies indicates that SeMet has weaker anticancer effects than most other forms of selenium. Recent studies showed that the anticancer activity of SeMet can be enhanced by methioninase (METase), indicating that SeMet metabolites are responsible for its anticancer activity. In the present study, we showed that wild-type p53-expressing LNCaP human prostate cancer cells were more sensitive to cotreatment with SeMet and METase than p53-null PC3 human prostate cancer cells. SeMet and METase cotreatment significantly increased levels of superoxide and apoptosis in LNCaP cells. Cotreatment with SeMet and METase resulted in increased levels of phosphorylated p53 (Ser15), total p53, Bax, and p21(Waf1) proteins. LNCaP cells treated with SeMet and METase also showed p53 translocation to mitochondria, decreased mitochondrial membrane potential, cytochrome c release into the cytosol, and activation of caspase-9. The effects of SeMet and METase were suppressed by pretreatment with a synthetic superoxide dismutase mimic or by knockdown of p53 via RNA interference. Reexpression of wild-type p53 in PC3 cells resulted in increases in superoxide production, apoptosis, and caspase-9 activity and a decrease in mitochondrial membrane potential following cotreatment with SeMet and METase. Our study shows that apoptosis induced by SeMet plus METase is superoxide mediated and p53 dependent via mitochondrial pathway(s). These results suggest that superoxide and p53 may play a role in cancer chemoprevention by selenium.