Both cancer and tumour-associated host cells are exposed to ionizing radiation when a tumour is subjected to radiotherapy. Macrophages frequently constitute the most abundant tumour-associated immune population, playing a role in tumour progression and response to therapy. The present work aimed to evaluate the importance of macrophage-cancer cell communication in the cellular response to radiation. To address this question, we established monocultures and indirect co-cultures of human monocyte-derived macrophages with RKO or SW1463 colorectal cancer cells, which exhibit higher and lower radiation sensitivity, respectively. Mono- and co-cultures were then irradiated with 5 cumulative doses, in a similar fractionated scheme to that used during cancer patients' treatment (2 Gy/fraction/day). Our results demonstrated that macrophages sensitize RKO to radiation-induced apoptosis, while protecting SW1463 cells. Additionally, the co-culture with macrophages increased the mRNA expression of metabolism- and survival-related genes more in SW1463 than in RKO. The presence of macrophages also upregulated glucose transporter 1 expression in irradiated SW1463, but not in RKO cells. In addition, the influence of cancer cells on the expression of pro- and anti-inflammatory macrophage markers, upon radiation exposure, was also evaluated. In the presence of RKO or SW1463, irradiated macrophages exhibit higher levels of pro-inflammatory TNF, IL6, CCL2 and CCR7, and of anti-inflammatory CCL18. However, RKO cells induce an increase of macrophage pro-inflammatory IL1B, while SW1463 cells promote higher pro-inflammatory CXCL8 and CD80, and also anti-inflammatory VCAN and IL10 levels. Thus, our data demonstrated that macrophages and cancer cells mutually influence their response to radiation. Notably, conditioned medium from irradiated co-cultures increased non-irradiated RKO cell migration and invasion and did not impact on angiogenesis in a chicken embryo chorioallantoic membrane assay. Overall, the establishment of primary human macrophage-cancer cell co-cultures revealed an intricate cell communication in response to ionizing radiation, which should be considered when developing therapies adjuvant to radiotherapy.