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Nanoplastics (NPs), as emerging contaminants, have attracted increasing attention for their effects on human exposure and potential health risks. The protein corona formed on the surface of NPs affects the biological activity and fate of the NPs in vivo. However, how environmental aging, an inevitable process once NPs enter the environment, affects the formation of protein corona on NPs is still unclear. This study investigated the changes in the compositions of protein corona formed on photo-aged polystyrene (PS) NPs in human bronchoalveolar lavage fluid (BALF), corresponding to the inhalation exposure pathway. The results demonstrated that both the species and abundance of proteins in the BALF protein corona on the surface of PS NPs were altered by aging. In addition, the aged PS NPs are more hydrophilic and less electronegative than the pristine PS NPs; hence, there is an increased sorption of more negatively charged hydrophilic proteins. Moreover, aging-induced alterations in BALF protein corona enhanced the uptake of aged PS NPs by lung macrophages J774A.1 through phagocytosis and clathrin-mediated endocytosis. These findings highlight the importance of environmental aging processes in the biosafety assessment of nanoplastics.