Cadmium (Cd) is a heavy metal, which will pollute the soil and affect the growth and development of plants. Grafting can alleviate the toxicity of heavy metals. Pumpkin (Cucurbita moschata Duch.) is the commonly-used grafting rootstock for melon plants, and the breeding of rootstock with cadmium tolerance plays a vital role in the safe production of melon plants. In this study, the rootstock pumpkin MB3, FB1 and their hybrid F1 ZF1 were used as experimental materials. The substrate culture was applied under conditions of cadmium (8, 16, and 24 mg/L), and the defense response of pumpkin rootstock to cadmium was studied, cadmium free stress was used as control. The experimental results indicated that under the cadmium stress, the growth of pumpkin seedlings was inhibited, but compared with the control, Cd promoted the plant height of ZF1. The determination of cadmium content in each part of pumpkin seedlings showed that the Cd accumulation ability of ZF1 roots was higher than that of parents, and the transfer coefficient to shoot was low, which reduced the concentration of Cd in leaves, improved the photosynthetic assimilation of leaves and Cd tolerance of pumpkin seedlings, and enhanced the activities of antioxidant enzymes (SOD, POD, CAT). Root activity and root scanner showed that ZF1 roots had the least damage under Cd stress compared with MB3 and FB1 parents. The distribution of cadmium concentration in root systems subcellular showed that under cadmium stress, ZF1 initiated the activity of membrane protective enzyme, relieved the damage to membrane, and reduced the toxicity of cadmium through the accumulation of cadmium in the cell wall that blocked its entrance to the cytoplasm. This study provides a theoretical foundation to breed cadmium-tolerant rootstocks for melon plants.