Kaempferol is a kind of natural flavonoid in many edible plants and reportedly has various physiological effects. In the present study, we conducted the genotoxicity (in vitro and in vivo) and 13-week subchronic toxicity studies of a new product, a kaempferol aglycone-rich food produced from enzyme-treated horseradish leaves, to evaluate its safety. In the bacterial reverse mutation test, the kaempferol aglycone-rich product showed positive results in some Salmonella typhimurium strains in the presence or absence of metabolic activation as well as other flavonoids. However, it did not increase micronucleated polychromatic erythrocytes taken from male Sprague−Dawley (SD) rats administered orally by gavage up to 4000 mg/kg for 2 consecutive days. In the 13-week subchronic toxicity study in SD rats, the kaempferol aglycone-rich product was orally administered by gavage once daily to SD rats for 13 weeks (91 days) at a dose of 500, 1000, or 2000 mg/kg/day. No toxic changes were observed at up to 2000 mg/kg/day. In conclusion, these findings indicated that the kaempferol aglycone-rich product was not genotoxic in vivo. The no-observed-adverse-effect level for both male and female rats was 2000 mg/kg/day, the highest dose tested, in the 13-week subchronic toxicity study in rats, suggesting it is safe for use as a food.