Macrophages are involved in many key physiological processes and complex responses such as inflammatory, immunological, infectious and neoplastic diseases. The appearance and activation of macrophages are thought to be rapid events in the development of many pathological lesions, including malignant tumours, atherosclerotic plaques, and arthritic joints. This has prompted recent attempts to use macrophages as novel cellular vehicles for gene therapy, in which macrophages are genetically modified ex vivo and then reintroduced into the body with the hope that a proportion will then home to the diseased site. Here, we describe a protocol for preparing monocyte-derived macrophages (MDM) and arming these with oncolytic viruses (OV) as a novel way for delivering anti-cancer therapies. In this approach, proliferation of macrophages co-transduced with a hypoxia-regulated E1A/B construct and an E1A-dependent oncolytic adenovirus, is restricted to prostate tumour cells using prostate-specific promoter elements from the TARP, PSA, and PMSA genes (Muthana et al., 2013; Muthana et al., 2011). When such co-transduced cells reach an area of extreme hypoxia (like that found in tumours), the E1A/B proteins are expressed, thereby activating replication of the adenovirus. The virus is subsequently released by the host macrophage and infects neighboring tumour cells. The virus then infects neighboring cells but only proliferates and is cytotoxic in prostate tumour cells. OV kill cancer cells by a number of mechanisms, including direct lysis, apoptosis, autophagy and shutdown of protein synthesis, as well as the induction of anti-tumoural immunity. Using macrophages to deliver OV ensures that they are protected from the many hazards they face in circulation including neutralizing antibodies, complement activation and non-specific uptake by other tissues such as the liver and spleen.