p66 Shc, an isoform of Shc adaptor proteins, is shown to mediate various signals, including cellular stress. However, little is known about its involvement in carcinogenesis. We previously showed that p66 Shc protein level is upregulated by steroid hormones in human carcinoma cells and is higher in prostate cancer (PCa) specimens than adjacent noncancerous cells. In this study, we investigated the role of p66 Shc protein in PCa cell proliferation. Among different PCa cell lines tested, p66 Shc protein level showed positive correlation with cell proliferation, that is, rapid-growing cells expressed higher p66 Shc protein than slow-growing cells. Exposure of slow-growing LNCaP C-33 cells to epidermal growth factor (EGF) and 5α-dihydrotestosterone (DHT) led to upregulation of proliferation and p66 Shc protein level. Conversely, growth suppression of fast-growing cells by cellular form of prostatic acid phosphatase (cPAcP) expression, a negative growth regulator, downregulated their p66 Shc protein level. Additionally, increased expression of p66 Shc protein by cDNA transfection in LNCaP C-33 cells resulted in increased cell proliferation. Cell cycle analyses showed higher percentage of p66 Shc-overexpressing cells at S phase (24%) than control cells (17%), correlating with their growth rates. On the other hand, transient knock-down of p66 Shc expression by RNAi in rapidly growing cells decreased their proliferation as evidenced by the reduced cell growth as well as S phase in p66 Shc-knocked down cells. The p66 Shc signaling in cell growth regulation is apparently mediated by extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK). Thus, our results indicate a novel role for p66 Shc in prostate carcinogenesis, in part, promoting cell proliferation.