Evaluation of biomaterial properties using THP-1 cells require the establishment of standardized protocols. Two potential methods are available, which differ in the timing of cell contact with the material; i.e. cell differentiation occurring simultaneous with or prior to polarization. No reports have examined the activation state of macrophages during differentiation. The aim of this study was to clarify the effects of biomaterials on THP-1 cells during differentiation. THP-1 cells were seeded on polymeric materials in the absence and presence of phorbol-12-myristate-13-acetate, and M1/M2 polarization was induced. The differentiation from THP-1 cells into macrophages was evaluated by loss of proliferation and acquisition of adhesion. The activation levels and M1/M2 polarization of M0 were assessed by IL-1β and MRC1 mRNA expression. Undifferentiated THP-1 cells were not markedly stimulated by interaction with biomaterials. However, THP-1 cells seeded on all the test materials differentiated into macrophages, and the macrophages polarized into different activated states depending on the material. These findings revealed the effects of material stimulation on macrophage activation state during differentiation. These results suggest that the step of cell differentiation and the step of contact with the material should be separated during systematic evaluation of biomaterials.