Few options are available for treating patients with advanced prostate cancer (PC). As PC is a slow growing disease and accessible by ultrasound, gene therapy could provide a viable option for this neoplasm. Conditionally replication‐competent adenoviruses (CRCAs) represent potentially useful reagents for treating PC. We previously constructed a CRCA, cancer terminator virus (CTV), which showed efficacy both in vitro and in vivo for PC. The CTV was generated on a serotype 5‐background (Ad.5‐CTV) with infectivity depending on Coxsackie‐Adenovirus Receptors (CARs). CARs are frequently reduced in many tumor types, including PCs thereby limiting effective Ad‐mediated therapy. Using serotype chimerism, a novel CTV (Ad.5/3‐CTV) was created by replacing the Ad.5 fiber knob with the Ad.3 fiber knob thereby facilitating infection in a CAR‐independent manner. We evaluated Ad.5/3‐CTV in comparison with Ad.5‐CTV in low CAR human PC cells, demonstrating higher efficiency in inhibiting cell viability in vitro. Moreover, Ad.5/3‐CTV potently suppressed in vivo tumor growth in a nude mouse xenograft model and in a spontaneously induced PC that develops in Hi‐myc transgenic mice. Considering the significant responses in a Phase I clinical trial of a non‐replicating Ad.5‐mda‐7 in advanced cancers, Ad.5/3‐CTV may exert improved therapeutic benefit in a clinical setting. J. Cell. Physiol. 229: 34–43, 2014. © 2013 Wiley Periodicals, Inc.