Selective replication-competent adenovirus serotype 5 vectors have been used for prostate cancer therapy. Unfortunately, gene transfer is inefficient because hormone-refractory metastatic prostate cancer cells have minimal coxsackievirus-adenovirus receptor expression. Vectors based on species B adenoviruses are attractive tools for use in human gene therapy because the viruses have low seroprevalence and they have efficient transduction capacity. Most species B adenoviruses use ubiquitously expressed complement-regulatory CD46 protein as a cellular receptor. Here we report the transduction efficacy and oncolytic capacity of a replication-competent Ad11p (RCAd11p) vector in human prostate cancer cells. Green fluorescent protein was efficiently expressed in a dose-dependent manner in PC-3 and DU 145 cells derived from metastasis of prostate cancer to bone and brain, respectively. However, transduction was less effective in LNCaP cells derived from prostate cancer metastasis to lymph nodes. The oncolytic capacity of the RCAd11p vector was 100 times higher in PC-3 cells than in the two other cell lines. The oncolysis was independent of the level of expression of p53 in the cells or on the absence of E1B55k expression in the vector. In vivo experiments revealed significant growth inhibition of PC-3 tumors in the xenograft mouse group treated with RCAd11p vector or Ad11pwt in comparison with the untreated control group. Thus, we have demonstrated that RCAd11p vector intrinsically possesses oncolytic properties, which were active in targeting tumor cells. Consequently, the novel RCAd11p vector has great potential for the treatment of incurable metastatic prostate disease.