Oncolytic viruses (OVs) are promising alternative biological agents for treating cancer. However, triggered immune responses against viruses and their delivery to tumor sites are their primary limitations in cancer therapy. To address these challenges, mesenchymal stem cells (MSCs) can serve as permissive tools for OVs loading and delivery to tumor sites. Here, we evaluated the in vitro and in vivo antitumor capability of adipose-derived mesenchymal stem cells (AD-MSCs) as a new vehicle for Dearing strain of reovirus (ReoT3D) loading. We first isolated and confirmed the purity of MSCs, and the optimized dose of ReoT3D for MSCs loading was computed by a standard assay. Next, we used murine CT26 cell line to establish the colorectal cancer model in BALB/c mice and demonstrated the antitumor effects of MSCs loaded with reovirus. Our results demonstrated that multiplicity of infection (MOI) 1 pfu/cells of reovirus was the safe dose for loading into purified MSCs. Moreover, our anticancer experiments exhibited that treatment with MSCs loaded with ReoT3D was more effective than ReoT3D and MSCs alone. Higher anticancer impact of MSCs loaded with OV was associated with induction of apoptosis, cell cycle arrests, P53 expression in tumor sections, and reduced tumor growth and size. The present results suggest that MSCs as a permissive shuttle for oncolytic virus (OV) delivery increased the anticancer activity of ReoT3D in mice models of colorectal cancer and these findings should be supported by more preclinical and clinical studies.