Protein expression of p63 is used to differentiate prostate cancer from benign mimickers. Recent studies suggest that it may also distinguish aggressive prostate cancer with down-regulated expression occurring in men with more advanced disease. We conducted a prospective study among 298 men ages 51 to 84 years who were diagnosed with prostate cancer in the Physicians' Health Study in 1983 to 2004 and whose tissue was available for immunohistochemical staining. We used Cox proportional hazards regression to evaluate the association of p63 protein expression with fatal prostate cancer. We correlated p63 expression with tumor cell proliferation (Ki-67) and apoptosis (TUNEL staining). The predominant location of tumor p63 staining occurred in the cytoplasm, an uncommon departure from the strong nuclear staining usually observed in nonneoplastic basal cells. Increasing expression of cytoplasmic p63 (tertiles) was associated with prostate cancer mortality (n = 19 deaths); the hazard ratios (95% confidence intervals) were 1.0 (reference), 4.0 (0.9-18.9), and 5.9 (1.3-27.5; P(trend) = 0.03). The positive trend remained significant (P = 0.047) after multivariable adjustment for age, year of diagnosis, and Gleason score. Higher tertiles of cytoplasmic p63 were also associated with reduced levels of apoptosis (P(trend) = 0.0408) and increased cellular proliferation (P(trend) = 0.0026). We found aberrant expression of p63 in the cytoplasm to be associated with increased prostate cancer-specific mortality up to 20 years after diagnosis. The mislocalized expression was associated with reduced apoptosis and higher proliferative activity and may suggest an oncogenic role in prostate cancer progression and survival.