It has been widely shown that biomaterial surface topography can modulate host immune response, but a fundamental understanding of how diﬀerent topographies contribute to pro-inﬂammatory or anti-inﬂammatory responses is still lacking. To investigate the impact of surface topography on immune response, we undertook a systematic approach by analyzing immune response to eight grades of medical grade polyurethane of increasing surface roughness in three in vitro models of the human immune system. Polyurethane specimens were produced with deﬁned roughness values by injection molding according to the VDI 3400 industrial standard. Specimens ranged from 0.1 μm to 18μm in average roughness (Ra), which was conﬁrmed by confocal scanning microscopy. Immunological responses were assessed with THP-1-derived macrophages, human peripheral blood mononuclear cells (PBMCs), and whole blood following culture on polyurethane specimens. As shown by the release of pro-inﬂammatory and anti-inﬂammatory cytokines in all three models, a mild immune response to polyurethane was observed, however, this was not associated with the degree of surface roughness. Likewise, the cell morphology (cell spreading, circularity, and elongation) in THP-1-derived macrophages and the expression of CD molecules in the PBMC model on T cells (HLA-DR and CD16), NK cells (HLA-DR), and monocytes (HLA-DR, CD16, CD86, and CD163) showed no inﬂuence of surface roughness. In summary, this study shows that modifying surface roughness in the micrometer range on polyurethane has no impact on the pro-inﬂammatory immune response. Therefore, we propose that such modiﬁcations do not aﬀect the immunocompatibility of polyurethane, thereby supporting the notion of polyurethane as a biocompatible material.