The effects of seawater stress on metabolism of reactive oxygen species (ROS) and chlorophyll (Chl) in chloroplast of two spinach cultivars, Yuanye spinach (seawater sensitive cultivar) and Helan No.3 (seawater tolerant cultivar) were investigated by hydroponics. The results showed that production rate of superoxide radical (O?2), content of hydrogen peroxide (H2O2) and malonaldehyde (MDA) in chloroplast of two spinach cultivars were remarkably increased by seawater with higher rate in cv. Yuanye; Under the stress of seawater, the activity of anti-oxidation enzyme such as superoxide dismutase (SOD), ascorbic acid peroxidase (APX) and glutathione reductase (GR), and the content of antioxidant such as ascorbic acid (AsA) and glutathione reduced (GSH) in chloroplast of cv. Yuanye were lower than those of cv. Helan No.3, while peroxidase (POD) activity in chloroplast of cv.Yuanye was higher than that of cv. Helan No.3; The supplement of methyl viologen (MV), the photo-oxidant, enhanced the production rate of O?2, the content of H2O2 and MDA in chloroplast of two spinach cultivars, while the addition of AsA, the eliminator for ROS, decreased ROS level and alleviated oxidization of plasmolemma. The content of chlorophyll b (Chlb), chlorophyll a (Chla) and precursor of Chl such as protochlorophyll (Pchl), Mg-protoporphyrinⅨ (Mg-ProtoIX), protoporphyrinⅨ(ProtoⅨ) and uroorphyrinogenⅢ (UroⅢ) was remarkably decreased, but the content of porphobilinogen (PBG) and δ-aminolevulinic acid (ALA) was increased by the stress of seawater, which lead to inhibition for Chl synthesis, and the inhibition was aggravated by MV and eliminated by AsA. Chlorophyllase (Chlase) activity in the leaves of cv. Yuanye was improved while it was not influenced in the leaves of Helan No.3 under seawater stress. The Chlase activity in the leaves of cv. Yuanye was more greatly affected by MV treatment than that of Helan No.3, however, Chlase activity of two cultivars was not influenced by AsA. These results suggest that ROS is closely related to Chl metabolism, which not only injure plasmolemma but also inhibit the process of transformation of PBG to UroⅢ as a result of Chl decomposition. In seawater tolerant cultivar (cv. Helan No.3), the ROS elimination is mainly depended on SOD and AsA-GSH system, which could alleviate oxidation injury of ROS to chloroplast membrane and inhibition of Chl synthesis, and Chlase activity was less influenced by seawater stress; while in seawater sensitive cultivar (cv. Yuanye), it is mainly depended on SOD and POD, which has limited ability to eliminate ROS and lead to accumulate mass ROS to severe oxidation injury of chloroplast membrane and inhibition of Chl synthesis, and Chlase activity was significantly improved by seawater stress, which enhanced Chl decomposition.