Internalization of nanoparticles (NPs), including nanoplastic, is one of the key factors determining their toxicity. In this work, we studied the toxicity and mechanisms of the uptake of model fluorescent polystyrene NPs (PS NPs) of three different sizes (30, 50, and 100 nm) in three human cancer cells lines; two originated from gut tissue (HT-29 and Caco-2) and one originated from liver tissue (Hep G2). Toxicity was measured by Neutral Red Assay (NRU), whereas mechanisms of uptake were studied using flow cytometry and different uptake inhibitors. The toxicity of the studied NPs followed a general rule observed for NPs-the smaller ones were more toxic than the larger ones. This relationship was dose dependent; however, the overall toxicity of the studied NPs was very low, despite the significant uptake of PS NPs. Although clathrin- and caveolin-dependent uptake is generally accepted as a major route of NP uptake, the inhibition of both mechanisms did not affect PS NP uptake in the cell lines studied in this work. Further experiments revealed that the major route of PS NP uptake in these cells is a scavenger receptor-mediated uptake.