Nanoplastics and microplastics (MPs) can significantly affect marine ecosystems and pose potential risks to human health. Although adverse effects stemming from direct exposure to MPs have been demonstrated at the cellular level in animal models, the potential toxicity of these materials in the human body remains uncertain. In this study, we investigated the three-dimensional (3D) behavior of dermal-derived cells exposed to MPs using artificially manufactured spherical primary polystyrene (PS) particles. To explore these effects, we used cellular spheroids as a 3D cell culture model, examined the size-dependent penetration of PS-MPs, and observed morphological alterations in the spheroids. Furthermore, we assessed changes in physiological activities, including reactive oxygen species, adenosine triphosphate, and lactate dehydrogenase, to elucidate the potential intra- and extracellular toxic reactions to PS-MPs. Additionally, our examination of cell-cell junctions and the extracellular matrix (ECM), along with analysis of the regulators involved in their decreased integrity, revealed negatively influenced changes in expression. This exposure study using spheroid models provides new insights into the potential toxicity of short-term exposure to MPs under conditions that closely resemble in vivo systems.