The composition of glycoalkaloids in tomato fruit changes with ripening. However, it has not been clarified whether the accumulation of glycoalkaloids is controlled by the ripening-inducing phytohormone, ethylene. Here, we report the effect of ethylene on the accumulation of tomato fruit glycoalkaloids. We investigated the effect of exogenously applied ethylene. In response to ethylene treatment, the content of alpha-tomatine decreased, whereas the content of esculeoside A increased. Next, we analyzed the fruits of ripening mutants, rin, nor, and Nr. In fruits of these mutant lines, the level of accumulation of esculeoside A decreased, whereas alpha-tomatine accumulated to higher levels than in wild-type fruit. These results demonstrated that the esculeoside A accumulation was associated with production and perception of ethylene. Additionally, the accumulation profiles of the intermediate metabolites of esculeoside A biosynthesis in ripening mutant fruits suggest that a glycosylation step in the putative pathway from alpha-tomatine to esculeoside A depends on ethylene.