This study investigates the effects of surface ligand distribution pattern (ligand clustering and density) on the internalisation of nanoparticles by a bronchial epithelial in vitro model (Calu-3 cells cultured as polarised layers). Control of ligand clustering and its surface density was achieved through the use of ovalbumin as an intermediate species to anchor the ligand to the nanoparticle surface. The model particulate system consisted of polystyrene nanoparticles surface-decorated via the adsorption of ovalbumin with conjugated folate ligand. The density of the displayed ligand was manipulated by controlling the conjugation level of folate to ovalbumin, while ligand clustering was achieved by co-adsorption of varying mixtures of folate-ovalbumin conjugate (at different ligand density levels) and unconjugated ovalbumin. Increasing overall ligand density on the nanoparticle surface resulted in increased internalisation of modified nanoparticles by the cells, up to a saturation level. Surface ligand density also affected the cellular uptake pathway; from predominantly clathrin to predominantly caveolae-mediated as the ligand density is increased. We further demonstrate that surface clustering of the folate ligand enhances cellular internalisation of nanoparticles, relative to its dispersed surface distribution. Our work suggests a simple way to prepare a model system where surface manipulations of ligand density and its distribution are possible and which can be used to study nanoparticle–cell interaction processes.