Monodisperse polypyrrole (PPy) nanoparticles with five different diameters (20, 40, 60, 80, and 100 nm) were fabricated via chemical oxidation polymerization in order to evaluate size-dependent cytotoxicity. The cellular uptake of PPy nanoparticles in human lung fibroblasts (IMR90) and mouse alveolar macrophages (J774A.1) was observed by transmission electron microscopy. The nanoparticles were internalized into the IMR90 via endocytosis. In the J774A.1, the nanoparticles were entered via phagocytosis and endocytosis. Endocytosed nanoparticles were transported to lysosome via endosome-network. The cytotoxicity and innate immune response of PPy-treated cells were systematically investigated by viability assay, oxidative stress, apoptosis/necrosis, and expression of costimulatory molecules. The viability, oxidative stress, and apoptosis/necrosis of PPy-treated cells revealed size- and dose-dependency. Because of phagocytosis, PPy treatment had more adverse effects on the J774A.1 than the IMR90. Innate immune response of PPy-treated macrophages was measured by the expression of costimulatory molecules on surface of the cells. The expression of costimulatory molecules involved in Th1 response (CD40 and CD80) was lightly up-regulated and the other costimulatory molecule related in Th2 response (CD86) was less expressed than a negative control. These findings may provide better nanotoxicological information of polymer nanomaterials, and support the further development of PPy nanoparticles in bioelectronic applications.