As the lung is one of the main routes of exposure to manufactured nanoparticles, we developed an in vitro model resembling the alveolo-capillary barrier for the study of nanoparticle translocation. In order to provide a relevant and ethical in vitro model, cost effective and easy-to-implement human cell lines were used. Pulmonary epithelial cells (Calu-3 cell line) and macrophages (THP-1 differentiated cells) were cultivated on the apical side and pulmonary endothelial cells (HPMEC-ST1.6R cell line) on the basal side of a microporous polyester membrane (Transwell®). Translocation of non-functionalized (51 and 110 nm) and aminated (52 nm) fluorescent polystyrene (PS) nanobeads was studied in this system. The use of Calu-3 cells allowed high transepithelial electrical resistance (TEER) values (>1000 Ω cm2) in co-cultures with or without macrophages. After 24 h of exposure to non-cytotoxic concentrations of non-functionalized PS nanobeads, the relative TEER values (%/t0) were significantly decreased in co-cultures. Epithelial cells and macrophages were able to internalize PS nanobeads. Regarding translocation, Transwell® membranes per se limit the passage of nanoparticles between apical and basal side. However, small non-functionalized PS nanobeads (51 nm) were able to translocate as they were detected in the basal side of co-cultures. Altogether, these results show that this co-culture model present good barrier properties allowing the study of nanoparticle translocation but research effort need to be done to improve the neutrality of the porous membrane delimitating apical and basal sides of the model.