Abstract: Royal jelly (RJ), a natural product secreted by honeybees, is used in various topical products for skincare and aids in maintaining cutaneous homeostasis. However, the mechanism underlying the effect of RJ on the skin has not yet been fully explored. Our previous data indicated that the epidermal equivalents become thicker and contain more p63-expressing proliferative cells after the addition of RJ to the medium. Therefore, we examined the effect of RJ on the proliferative ability of human primary epidermal keratinocytes (HPEKs) in a two-dimensional culture here. We observed only a slight increase in the proliferation of cells with the addition of RJ, suggesting that it is not actively involved in the proliferation of HPEKs. However, population doubling was enhanced in the RJ-treated group in the long-term culture experiment, indicating that RJ inhibits senescence. Additionally, RJ suppressed cellular senescence by regulating the expression levels of ΔNp63, p16, and p21. These results were further confirmed by the presence of major fatty acids, such as 10-hydroxy-2-decenoic acid, in RJ. Overall, our data indicate that RJ can maintain epidermal stem cell properties by repressing senescence.