Abstract: With indoor static simulation test, this paper studied the effects of different concentrations (0.01, 0.02, 0.05, 0.07 and 0.1 mg·L^-1) pyrene on the pyrene accumulation, free radical, antioxidant defenses, and the contents of chlorophyll and soluble sugar in Vallisneria spiralis. The results showed that after 10 days exposure to pyrene, V. spiralis leaf could accumulate large amount of pyrene. The free radical generation, peroxidase (POD) activity and malondialdehyde (MDA) content had a persistent increase, but the increment decreased when exposed to higher concentrations (>0.05 mg·L^-1) pyrene. Glutathione S-transferase (GST) activity and oxidized glutathione (GSSG) and soluble sugar contents increased with increasing concentration of pyrene, while reduced glutathione (GSH) and chlorophyll contents were in adverse. It was concluded that V. spiralis was sensitive to pyrene, and the stress effect would happen at 0.01 mg·L^-1 of pyrene.