Several studies have evaluated the efficacy of using human oncolytic adenovirus-loaded mesenchymal stem cells for cancer treatment. For example, we have described the antitumor efficacy of CELYVIR, autologous bone marrow mesenchymal stem cells infected with the oncolytic adenovirus ICOVIR-5, for treatment of neuroblastoma patients. Results from this clinical trial point out the role of the immune system in the clinical outcome. In this context, a better understanding of the immunophenotypic changes of human mesenchymal stem cells upon adenoviral infection and how these changes affect human autologous or allogeneic peripheral blood mononuclear cells (PBMCs) could guide strategies to improve the antitumor efficacy of infected Mesenchymal Stem Cells (MSCs). In this work, we show how infection by an oncolytic adenovirus (OAdv) induces Toll-like receptor 9 overexpression and activation of the NF-κB pathway in menstrual blood-derived mesenchymal stem cells (MenSCs), leading to a specific cytokine secretion profile. Moreover, a pro-inflammatory environment, mainly mediated by monocyte activation that leads to the activation of both T-cells and natural killer cells (NK cells), is generated when OAdv-loaded MenSCs are co-cultured with allogeneic PBMCs. This combination of allogeneic PBMCs and OAdv-loaded MenSCs enhances antitumor efficacy both in vitro and in vivo, an effect partially mediated monocytes and NK cells. Altogether our results demonstrate not only the importance of the immune system for the oncolytic adenovirus-loaded MSCs antitumor efficacy, but in particular the benefits of using allogeneic MSCs for this therapy.