Populus is one of the most important tree species in the economic plantations in China. However, poplar plantations are vulnerable to the continuous monoculture problems. Previous studies have focused on low availability of soil nutrients and poor management as the possible causes for such decline. With the progress of chemical ecology, allelopathy and autotoxicity in forest plantations have been increasingly regarded as key triggers of the problems associated with continuous monoculture. To the best of our knowledge, few studies on poplar autotoxicity have been based on long-term samples, even though poplar monoculture is common in planting practice. Moreover, experiments to compare the effects of low availability of soil nutrients and autotoxicity have not been attempted. In this study, we selected soil samples from three generations of poplar plantations, which included monoculture plantation as old as 40 years, and carried out systemic germination bioassays of lactuca sativaseeds and seedling growth tests on branch cuttings of poplar trees grown on soils in which successive generations of the plant had been grown. We identified the inhibitory activity of rhizosphere soil and the effects of concentration dependence and independence on nutrient inhibitory activity, which provided basic experimental evidence for further study on autotoxicity in continuously cropped poplar plantations. In a laboratory experiment, we contrasted the inhibitory activities of rhizosphere soil and non-rhizosphere soil on continuously cropped poplar plantations by determining lettuce seed germination in the soil extracts. The results showed that rhizosphere soil significantly inhibited lettuce seed germination compared to the non-rhizosphere soil. Concentration-controlled assays of rhizosphere soil extracts of generations I, Ⅱ, and Ⅲ (GenI, GenⅡ, and GenⅢ) poplar plantations were used to investigate the inhibitory dose attribution effect. The results showed that greater the soil extract concentration, stronger was the inhibitory effect on seed germination, suggesting that soil extracts inhibited seed germination in a concentration-dependent manner. The results also showed that concentration dependence was strongest in GenⅢ poplar plantations. The rhizosphere soil extracts of GenI, GenⅡ, and GenⅢ poplar plantations were divided into two groups (an added-nutrition group and a non-nutrition group) of culture solutions, which determined the length of shoot and root in the poplar branch cuttings. The results showed that the length of shoot and root in the GenⅢ treatments were significantly lower than in the GenⅡ treatments, whereas those in the GenⅡ treatments were significantly lower than in the GenI treatments. There were no significant differences between the GenI treatments and the control group. This tendency toward inhibitory activity on plant growth increased with time (with the successive poplar generations) and was more prominent in the added-nutrition group. Our results, therefore, suggest that autotoxins accumulated through root exudates in the rhizosphere soil of continuously monocultured poplar plantations. As a result of concentration dependence, autotoxins did not significantly inhibit the plant growth at GenI stage. With increasing years of monoculture, the autotoxic effects significantly inhibited the plant growth by GenⅢ stage and were especially significant in the soils that received artificial nutrition. Therefore, long-term monoculture practice and inhibitory autotoxicity of rhizosphere soil are likely to cause the continuous planting problems seen in poplar plantations.