The concern about titanium dioxide nanoparticles (TiO-NPs) toxicity and their possible harmful effects on human health has increased. Their biological impact is related to some key physicochemical properties, that is, particle size, charge, crystallinity, shape, and agglomeration state. However, the understanding of the influence of such features on TiO-NP toxicity remains quite limited. In this study, cytotoxicity, proinflammatory response, and oxidative stress caused by five types of TiO-NPs with different physicochemical properties were investigated on A549 cells used either as monoculture or in co-culture with macrophages differentiated from the human monocytic THP-1 cells. We tailored bulk and surface TiO physicochemical properties and differentiated NPs for size/specific surface area, shape, agglomeration state, and surface functionalization/charge (aminopropyltriethoxysilane). An impact on the cytotoxicity and to a lesser extent on the proinflammatory responses depending on cell type was observed, namely, smaller, large-agglomerated TiO-NPs were shown to be less toxic than P25, whereas rod-shaped TiO-NPs were found to be more toxic. Besides, the positively charged particle was slightly more toxic than the negatively charged one. Contrarily, TiO-NPs, whatever their physicochemical properties, did not induce significant ROS production in both cell systems compared to nontreated control groups. These results may contribute to a better understanding of TiO-NPs toxicity in relation with their physicochemical features.