Immune cell-based therapy is a promising approach for cancer immunotherapy. Macrophages can be used for this purpose if their tumoricidal activity and viability are properly controlled. In the present study, we aimed to enhance these properties of macrophages by constructing uniformly sized multicellular spheroids. Mouse macrophage-like J774.1 cells were selected as model macrophages, and poly(N-isopropylacrylamide)-coated polydimethylsiloxane-based microwell plates with an approximate diameter of 750 μm were used to prepare J774.1 spheroids. J774.1 spheroids were successfully generated, and the viability of cells in the spheroids was over 95%. J774.1 spheroids showed higher mRNA expression of induced nitric oxide synthase, a marker of M1-type activated macrophages, than monolayered J774.1 cells. The production of reactive oxygen species was also high in J774.1 spheroids, suggesting the existence of hypoxic regions in the spheroids. J774.1 spheroids released more tumor necrosis factor-α than monolayered cells upon stimulation with lipopolysaccharide. Moreover, J774.1 spheroids in the upper compartment of the Transwell system more efficiently inhibited the proliferation of mouse adenocarcinoma colon 26 cells in its lower compartment than monolayered J774.1 cells did. These results indicate that spheroid formation can be used to increase the tumoricidal activity of macrophages for use in cell-based cancer immunotherapy.