Oncolytic strains of vaccinia virus are currently in clinical development with clear evidence of safety and promising signs of efficacy. Addition of therapeutic genes to the viral genome may increase the therapeutic efficacy of vaccinia. We evaluated the therapeutic potential of vaccinia virus expressing the sodium iodide symporter (NIS) in prostate cancer models, combining oncolysis, external beam radiotherapy, and NIS-mediated radioiodide therapy. The NIS-expressing vaccinia virus, GLV-1h153, was tested in in vitro analyses of viral cell killing, combination with radiotherapy, NIS expression, cellular radioiodide uptake and apoptotic cell death in PC3, DU145, LNCaP and WPMY-1 human prostate cell lines. In vivo experiments were carried out in PC3 xenografts in CD1 nude mice to assess NIS expression and tumour radioiodide uptake. In addition, the therapeutic benefit of radioiodide treatment in combination with viral oncolysis and external beam radiotherapy was measured. In vitro viral cell killing of prostate cancers was dose- and time-dependent and was through apoptotic mechanisms. Importantly, combined virus therapy and ionising radiation did not adversely affect oncolysis. NIS gene expression in infected cells was functional and mediated uptake of radioiodide both in vitro and in vivo. Therapy experiments with both xenograft and immunocompetent TRAMP mouse models showed that the addition of radioiodide to VV-NIS-infected tumours was more effective than each single-agent therapy, restricting tumour growth and increasing survival. In conclusion, VV-NIS is effective in prostate cancer models. This treatment modality would be an attractive complement to existing clinical radiotherapy practice.Gene Therapy accepted article preview online, 27 January 2016. doi:10.1038/gt.2016.5.