In this study, the watermelon cultivar ‘Zaojia 8424’ and pumpkin cultivar ‘Weizhen No. 1’ were used as materials, and two grafting combinations including watermelon/pumpkin (W/P) and pumpkin/pumpkin (P/P), were set up, using the onecotyledon splice grafting method. The pumpkin seedlings without grafting (P) were used as control. The effects of watermelon scion on root growth and sugar metabolism of grafted seedlings were measured. The results showed that: (1) in W/P, the watermelon scion caused significantly lower root growth index (fresh quality, dry mass, and root volume), root vigor, and shoot growth index (fresh quality, dry mass, and leaf area) of the grafted seedlings than those of the P/P and P seedlings. (2) The contents of total sugar, soluble sugar, reducing sugar, starch, as well as sucrose, glucose and fructose in the roots increased in this order: W/P < P/P < P, and there were significant differences between the treatments at 18 d post grafting. (3) Compared to the P/P and P treatments, watermelon scion caused lower activity of cell wall invertase (CWIN) and hexokinase (HXK) in the roots of W/P grafted seedlings at 18 d post grafting, and accordingly, the expression levels of CWIN and HXK were significantly reduced. The results indicated that watermelon scion could repress the sugar accumulation in the roots of grafted seedlings and inhibit the enzymatic activities of CWIN and HXK, and thus suppress the root growth and vigor of grafted seedlings.