Ubiquitous nanoplastics (NPs) increase exposure risks to humans through the food chain and/or other ways. However, huge knowledge gaps exist regarding the fate and adverse impact of NPs on the human cardiovascular system. Autophagy is an important catabolic pathway that disposes of cytoplasmic waste through the lysosomes. In this study, we pursued to determine the interaction and autophagy effect of polystyrene nanoplastics (PS-NPs) (100 and 500 nm in size) on human umbilical vein endothelial cells (HUVECs). The results showed both sizes of PS-NPs interacted with almost all the treated HUVECs in a time- and concentration-dependent manner, and 500 nm PS-NPs were only bound to the surface of cell membranes, whereas 100 nm PS-NPs were taken up by HUVECs and aggregated in the cytoplasm. Furthermore, exposure to 25 μg/mL of 500 nm PS-NPs for 48 h significantly increased lactate dehydrogenase release from HUVECs, while internalized 100 nm PS-NPs not only caused cell membrane damage, but also induced autophagy initiation and autophagosome formation. By a mCherry-GFP-LC3 lentivirus infection assay, we also demonstrated that autophagic flux level was impaired in response to 100 nm PS-NPs. Herein, our results provide new insight into the size-dependent internalization and autophagy response to PS-NPs in HUVECs.