When radish chloroplasts were pretreated with 1 mM spermidine (Spd) and then exposed to 30 M paraquat (PQ), they improved their tolerance to subsequent PQ-induced oxidative damages. That included the decreases in the contents of chlorophyll, protein, and ascorbate, as well as the increases in malondialdehyde (MDA) and H2O2 levels. Analysis of antioxidant enzymes showed that Spd pretreatment effectively prevented the PQ-induced decreases in the total activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX). In contrast, the normally enhanced activities of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) in PQ-treated chloroplasts were reversed by Spd pretreatment. In a native gel assay, the Cu/ZnSOD isozyme, which disappeared under the PQ alone treatment, was significantly recovered when tissues were pretreated with Spd. The dominant APX4 isozyme activity, which was preferentially decreased in response to PQ alone treatment, was also strongly reactivated by earlier Spd exposure. Therefore, we suggest that Spd could play a substantial role in protecting the radish chloroplasts from PQ stress. Furthermore, the enhancement of the Cu/ZnSOD and APX4 isozymes by Spd pretreatment seems to be responsible for prevention of the PQ-induced decreases in the total activities of SOD and APX, thereby providing a tolerance to PQ toxicity.