Codonopilate A (1), a triterpenyl ester, was isolated from monocultivated soil of annual Codonopsis pilosula and identified as the main autotoxin. The yield ratio of codonopilate A in dried soil was calculated as 2.04 μg/g. Other two triterpenoids, taraxeryl acetate (2) and 24-methylenecycloartanol (3), were isolated and identified as well showing weaker autotoxity. This was the first time that the potential allelochemicals and autotoxins in the cultivated soil of Codonopsis pilosula were reported. Accumulation of reactive oxygen species (ROS) induced by the autotoxins in the root tips of Codonopsis pilosula was considered as an important factor for the phytotoxic effect. This work systematically investigates the allelopathic and autotoxic effect of Codonopsis pilosula, and the preliminary autotoxic action mode of the three autotoxins. These findings are helpful to understand the molecular mechanism of autotoxicity and conducive to explore proper ways to degrade the autotoxins and eliminate the replanting problems of Codonopsis pilosula.