INTRODUCTION: Natural killer (NK) cells are innate lymphocytes with a key role in the defense against tumors. Recently, allogeneic NK cell-based therapies have gained interest because of their ability to directly lyse tumor cells without inducing graft-versus-host disease. As NK cells are also able to influence the function of other immune cells (most notably dendritic cells (DC)), a better understanding of the effects of allogeneic NK cell products on the host immune system is required. In this study, we analyzed the effects of an allogeneic off-the-shelf NK cell product, on the tumor microenvironment (TME) of primary and metastatic colorectal cancer (pCRC and mCRC, respectively). Moreover, we explored if the combination of NK cells with R848, a toll-like receptors 7/8 ligand, could further enhance any pro-inflammatory effects. METHODS: Ex vivo expanded umbilical cord blood stem cell derived NK cells were co-cultured with pCRC or mCRC single-cell suspensions in the presence or absence of R848 for 5_days, during and after which flow cytometry and cytokine release profiling were performed. RESULTS: NK cells efficiently induced lysis of tumor cells in both pCRC and mCRC single-cell suspensions and thereby controlled growth rates during culture. They also induced differentiation of infiltrating monocytic cells to an activated DC phenotype. Importantly, this NK-mediated myeloid conversion was also apparent in cultures after tumor cell depletion and was further enhanced by combining NK cells with R848. Moreover, NK cells, and to a greater extent, the combination of NK cells and R848, triggered CD8(+) and CD4(+) T-cell activation as well as a reduction in activated regulatory T cell rates. Finally, the combination of NK cells and R848 induced a pro-inflammatory shift in the cytokine release profile resulting in higher levels of interferon (IFN)-_, interleukin (IL)-2, IL-12p70, and IFN-_ as well as a reduction in IL-6, in both pCRC and mCRC cultures. CONCLUSION: Allogeneic NK cells engaged in favorable myeloid crosstalk, displayed effective antitumor activity and, when combined with R848, induced a pro-inflammatory shift of the CRC TME. These findings prompt the investigation of NK cells and R848 as a combination therapy for solid tumors.