The discovery of plastic and metal nanoparticles in organisms, foods, and beverages has generated numerous studies on the effects of these particles on the barrier cells and their subsequent absorption into the body. Following ingestion, nanoparticles travel down the gastrointestinal tract (GIT), and their physicochemical characteristics change in response to the change in proteins and pH during their digestion. We measured the translocation of digested nanoparticles across a co-culture monolayer of Caco-2 and various combinations (1:9, 5:5, and 9:1) of HT29-MTX-E12. The in vitro model of the intestine was used to determine the translocation of digested 20 nm polymethacrylate (PMA) particles and the accompanying monolayer barrier effects after a 72 h exposure. The in vitro digestion increased the agglomeration and hydrodynamic diameters and decreased the surface charge of the nanoparticles. For NH2-functionalized polymethacrylate nanoparticles (PMA-NH2), the diameters increased from 57 nm (water) to 3800 nm (media), or 2660 nm (chyme). These nanoparticles compromised the integrity of the monolayer (trans-epithelial electrical resistance, Lucifer yellow translocation) and translocated across all the cell ratio configurations. Digestion can have a large effect on nanoparticle agglomeration and surface charge. Excess mucous was not seen as a barrier to the translocation of PMA-NH2.