Abstract: Microplastics pose a potential threat to marine ecosystems due to their small size and not readily degradable. Microplastics ingested by marine organisms can accumulate or transfer in different tissues and organs, such as the digestive tract, and cause a series of biological effects. However, current toxicological studies lack the consideration of natural environmental factors, such as the role of aging. In this study, the marine copepod Tigriopus japonicus was used as the test organism; we investigated the toxicokinetic process and antioxidant defense mechanism of T. japonicus under natural seawater colonization in response to 10 μm polystyrene (PS) microspheres (exposed at concentrations of 100 particles/mL and 1000 particles/mL). It was found that the colonized effect increased the uptake of PS microspheres by T. japonicus, and the uptake tended to increase and then decrease with the exposure time (3 h, 6 h, 12 h, 24 h, and 48 h). After 48 h of exposure, T. japonicus ingested 1.9 times more highly concentrated (1000 particles/mL) colonized PS microspheres (4.6 ± 0.5)/individual than pristine PS microspheres (2.5 ± 0.1)/individual. Colonization had almost no effect on the excretion process of PS microspheres in T. japonicus. The residual percentage of PS microspheres in T. japonicus remained relatively high (> 30%) after 48 h. Under the low concentration (100 particles/mL) exposure condition, colonized PS microspheres significantly induced superoxide dismutase (SOD) and glutathione S-transferase (GST) activities, up-regulated the expression of related genes (MnSOD, GST, GR, and GPx), inhibited catalase (CAT) activity and down-regulated the relative expression of CAT genes in T. japonicus compared with that of pristine PS microspheres, and higher concentrations of PS microspheres caused greater damage to the antioxidant defense system of T. japonicus. Overall, the colonization effect increased the uptake of PS microspheres by T. japonicus, which exacerbated the oxidative stress in T. japonicus caused by the colonized PS microspheres to a certain extent. The results of the study are informative for scientific assessment of the ecological risk of microplastics in real marine environments. Our findings contributed to the scientific ecological risk assessment of microplastics in the natural marine environment.