Knowledge of the tumor microenvironment is crucial for developing an effective strategy to treat cancer. Recently, anticancer therapies targeting macrophages have been intensively investigated. Increased understanding of the importance of the tumor microenvironment has led to the development of three-dimensional (3D) in vitro tumor models. However, established techniques for studying tumor-associated macrophages in vitro are limited. We have previously characterized a 3D breast cancer model consisting of breast cancer cells and fibroblasts cocultured on a silk scaffold. In the present study, the influence of this model on macrophage polarization was investigated. The expression of macrophage markers was studied using reverse transcription-quantitative PCR and flow cytometry. The activity of nitric oxide synthase and arginase in macrophages was also measured. The presented model appeared to induce the polarization of macrophages towards an M2 phenotype. In this 3D tumor model, the in vivo behavior of macrophages could be reproduced. This model may be beneficial for the study of tumor biology and for screening drugs.