Objective The purpose of this study was to investigate the effect of royal jelly ( RJ) on serum hormone levels and mammary gland tissues of female non-pregnant rats (late puberty to early sexual maturity). Methods Fifty Wistar rats were randomly divided into 5 groups with 10 rats in each group. Rats in the treatment group were given RJ at different doses (100, 200, 400, 800 mg / (kg·d)), while rats in the control group (CK group) were given sterile water ( 2 mL). After 35 d, the growth index and nipple diameter were measured. The serum levels of estrogen ( E2), progesterone ( P) and prolactin ( PRL) were measured by ELISA. After hematoxylin-eosin staining of breast tissue, histological morphological changes were observed. Results The result showed that serum E2 and PRL levels in the RJ200 group and RJ400 group were significantly higher than those in the CK group (P< 0. 05), while serum P levels in the RJ200, RJ400 and RJ800 groups were significantly lower than those in the CK group (P< 0. 05). Serum E2, P and PRL levels in the RJ100 and CK groups were not significantly different (P< 0. 05). Breast duct diameter measurements showed that compared with the CK group, duct diameter in each of the treatment groups was significantly increased (P< 0. 05). The histological changes in the breast were consistent with duct diameter in the RJ100 group being lower than that in the RJ200, RJ400 and RJ800 groups. In addition, the diameter of the nipple in the RJ200 and RJ800 groups was slightly increased compared with the CK group (P< 0. 05). Conclusions Daily intake of low-dose (100 mg / kg) RJ did not affect serum hormone levels or breast development in female non-pregnant rats. However, at higher doses of 200 and 400 mg / (kg ·d), it may adversely affect the development of breast tissue and the stability of serum hormone levels in female non- pregnant rats.