Breast cancer is the most commonly diagnosed type of cancer among the female population worldwide. It is a disease with a high incidence and geographic distribution that negatively impacts global public health and deleteriously affect the quality of life of cancer patients. Among the new approaches, cancer immunotherapy is the most promising trend in oncology by stimulating the host's own immune system to efficiently destroy cancer cells. Recent evidence has indicated that iron oxide nanoparticles can promote the reprograming of M2 into M1 macrophages with anti-tumor effects in the tumor microenvironment. Thus, the aim of the present work was to evaluate the ability of polyaniline-coated maghemite (Pani/γ-FeO) nanoparticles to modulate human macrophages in 2D monolayers and 3D multicellular breast cancer models. It was observed that Pani/γ-FeO NPs re-educated IL-10-stimulated macrophages towards a pro-inflammatory profile, decreasing the proportion of CD163 and increasing the CD86 proportion in 2D models. NPs were successfully taken-up by macrophages presented in the 3D model and were also able to induce an increasing in their CD86 proportion in triple MCTs model. Overall, our findings open new perspectives on the use of Pani/γ-FeO NPs as an immunomodulatory therapy for macrophage reprogramming towards an anti-tumor M1 phenotype, providing a new tool for breast cancer immunotherapies.