Abstract: Replant disease is a serious problem for the development of sustainable cropping systems of strawberry, Fragaria ananassa. Previous studies have shown that auto-toxicoids secreted from roots play an important role in replant disease of some crops, and autotoxicity of root exudates is a very important aspect of understanding replant disease mechanisms. In this paper, root exudates were extracted and their autotoxicity was studied by using tissue culture methods in order to avoid interferences from external factors. Root tissue culture mediums (MS+BA 0.2 mg·L-1), in which one generation of seedlings had been cultivated, were collected in a bio-clean cabinet and mixed with a new culture medium. Five treatments were set up in which the volume proportion of the new culture medium to the used culture medium were 10∶30, 15∶25, 20∶20, 25∶15 and 30∶10. Tissue culture seedlings of the same age and with the same appearance were chosen and inoculated in the five culture medium treatments. Our results showed that on the 40th day after inoculation, development and growth of roots were inhibited in mixed tissue culture mediums, and the inhibitory effects were greater as the proportion of the used culture medium increased. As a result, increments of plant height and leaves, number of roots, length of roots, and weight of plants were reduced in mixed culture mediums as compared to the pure new culture medium treatment. The vermiculite in which strawberry tissue culture seedlings had been cultivated for 60 days was collected and placed into 500 ml erlenmeyer flasks. The same quantity of distilled water was added to each flask and then shaken on a shaker for 48 hours. After centrifuging (2 670×g) for 20 minutes, the supernatant was collected and evaporated on a rotary vaporizer at 42 ℃ to 1/10 volume, and then filtered through qualitative filter paper. The extract was diluted in a series of density solutions of 50%, 40%, 30%, 20% and 10% (V%). Strawberry seedlings were treated once every 5 days with 10 ml of the different solutions and the growth of seedlings measured after 40 days. Control seedlings were treated with distilled water. Results showed that root exudates of strawberry could inhibit the growth of strawberry, and the inhibitory effect became stronger with increasing concentrations of root exudates. Physiological disease occurred in treatments treated by root exudates, and the disease index was greater as the concentration of root exudates increased. At the same time, strawberry root activity was also restrained by root exudates. Physiological analysis showed that activities of 2,3,5-Tripheyl Tetrazolium Chloride (TTC) reduced and superoxide-dismutase (SOD) decreased, and root relative conductivity and malondialdelyole (MDA) contents increased. The greater the concentration of root exudates, the more significant the effect on all of these physiological factors. Our results showed that when root exudates accumulated in their growing medium, the growth and metabolism of strawberry roots were inhibited, which resulted in an increase in the percentage of electrolytes in cells, a decrease in the free radical scavenging activity of roots, and an increase in root lipid peroxidation. Hence, the growth and development of strawberry seedlings showed reduced vigor, which could lead to declines in root resistance to soil-borne disease, and replant disease of strawberry could become increasingly serious.