Plastic pollution is a growing environmental and health issue due to the increasing presence of micro- and nanoplastics in terrestrial and aquatic ecosystems. Polystyrene nanoplastics (PS-NPs) are among the most extensively studied because of their wide occurrence, physicochemical stability, and availability for laboratory research. Their nanoscale size enables interaction with biological systems at the molecular level, promoting internalization, intracellular trafficking, and potential bioaccumulation. This review summarizes current knowledge on the cellular effects and molecular mechanisms of PS-NPs, particularly in human gastrointestinal models. The gastrointestinal tract is a primary route of nanoplastic exposure, where PS-NPs can cross epithelial barriers, interact with immune and epithelial cells, and disturb cellular homeostasis. Once internalized, PS-NPs can induce oxidative stress, mitochondrial dysfunction, and dysregulation of autophagy, leading to alterations in lipid and glucose metabolism. Excessive synthesis of reactive oxygen species may trigger DNA damage, activate the ATM/ATR–p53 signaling pathway, and impair DNA repair mechanisms, thereby contributing to genomic instability. Emerging evidence also shows that PS-NPs can interact with ion channels, affecting calcium homeostasis, membrane potential, and cell viability. Overall, these findings highlight the complex and multifaceted toxicity of PS-NPs at the cellular level and underscore the need for further research to assess the long-term risks of nanoplastic exposure.