Although it is proved that humans ingest microplastics via food, and microplastics were found in human tissues, blood and feces, there needs to be more data on the properties and health-related effects of plastic particles that interact with food and undergo digestion. This study aimed to examine the impact of a real food matrix, milk, on the behavior and gastrointestinal fate of polystyrene microparticles (PSMP). In the presence of the food matrix, the net negative ζ-potential values of PSMP (diameter size of 1.823 μm) decreased significantly due to the formation of the corona, mostly consisting of α and β-casein fragments. Protein corona profiles and morphologies of particles incubated with whole and skim milk were found to be similar, and the protein profiles were completely altered after in vitro digestion simulation. In vitro and in vivo toxicity studies showed that neither bare PSMP nor food-interacted PSMP pose acute toxicity on the Caco-2 cell line and zebrafish embryos under the chosen experimental conditions. In summary, these results may contribute to a better understanding of changes that microplastics undergo in foods. Further studies on repeated exposure or chronic toxicity are needed to fully reveal the effect of food matrix on microplastic toxicity.