By using the techniques of tissue culture, bio-assay and laboratory analysis, this paper studied the effects of the allelopathic chemicals from pumpkin (Cucurbita moschata) roots on the seed germination and seedling growth of pumpkin, wheat (Triticum aestivum), and radish (Raphanus sativus). The pumpkin root was cultured on a sterile B5 media, and the concentrations of macro- and microelements, organic supplements and hormones in the media were adjusted by using an orthogonal design. After culturing, the culture media was filtered and used in a bioassay to test the autotoxicity and allelopathic effects. The results showed that the pumpkin had both autotoxic and allelopathic effects, and the media having been used to culture the pumpkin roots contained the chemicals that significantly inhibited the seedling growth of wheat and radish. The allelopathic effect decreased when the culture media was diluted. The production of allelochemicals seemed to be related to the growth rate of the pumpkin roots. When the root growth was rapid, the concentration of allelochemicals was high. The allelopathic effect was stronger on radish than on wheat. The optimum concentrations of macro- and microelements, vitamins and hormones for culturing pumpkin root were determined, and the effect of pumpkin root nutrition on the production of allelochemicals was tested. The results indicated that pumpkin root nutrition had a significant effect on the production of allelochemicals.