The production of melanin is not only activated by external factors such as sunlight or UV-exposure, but is also considered to be triggered by hormonal factors, particularly sex hormones such as ovarian hormones. Previously, keishibukuryoganryokayokuinin (KBY) was reported to increase the pigmentation and moisture content of dermis in women during the luteal phase of the menstrual cycle, thus suggesting that progesterone could play a critical role in the development of skin pigmentation. In the present study, female DBA/2 mice, a dilute brown strain, were used to examine the effects of KBY on the increase in epidermal pigment cells in mice exposed to ultraviolet B (UVB) radiation or progesterone in an attempt to elucidate its mechanism. An increase in epidermal pigment cells was observed in mice exposed to progesterone. To the best of our knowledge, this is the first study to demonstrate that progesterone causes pigmentation in vivo. Furthermore, administration of KBY to progesterone-exposed mice significantly reduced the number of epidermal pigment cells. However, KBY had no such effects on UVB-induced pigmentation. Another important finding was the gain in body weight in progesterone-exposed mice, while body weight gain was reduced by KBY. The body weight gain was believed to be due to sodium and fluid retention, a kind of adverse effect of progesterone, which may further affect the intracellular pH of melanosomes, which synthesize melanin, in turn, leading to melanin production because tyrosinase activity is linked to the intracellular pH environment. This may help explain the mechanism of the role of KBY in pigmentation.