[Objectives] The study aims to explore the effects of exogenous gonadotropin on epidermal growth factor (EGF) expression level and action characteristics in Hyla Rabbit’s (Oryctolagus cuniculu) reproductive organs. [Methods] In this experiment, 30 Hyla Rabbits (6 male and 24 female) were randomly selected. Female rabbits were randomly divided into superovulation group (intramuscular injection of 70 U pregnant mare serum gonadotropin +100 U human chorionic gonadotropin, n = 12) and control group (no injection of any hormone, n = 12). And Real-time fluorescence quantitative PCR (qRT-PCR), Western-blot and immunohistochemistry were used to study the expression and localization of epidermal growth factor (EGF) in ovaries, oviduct and uterus in superovulation group. SPSS 25.0 software was used for independent sample t test analysis of relevant data. Results were expressed as “mean ± standard deviation”, P < 0.01 means significant difference, P < 0.05 means significant difference. [Results] The results showed that the relative expression level of epidermal growth factor mRNA in different tissues was significantly higher in the superovulation group than in the control group (uterus:t = 17.053, P < 0.01; oviduct:t = 3.009, P < 0.01; ovaries:t = 14.125, P < 0.01) (Fig.1). The relative expression of EGF protein was also significantly higher in different tissues of the superovulation group than the control group (uterus:t = 20.663, P < 0.01; oviduct:t = 19.537, P < 0.01; ovaries:t = 21.272, P < 0.01) (Fig.2). And the positive signals were located in primary oocytes, granulosa cells, endometrium cells, mesenchymal cells and vascular endothelial cells respectively (Fig. 3). Oviduct ciliated cells, basal cells, mucosal epithelium, muscularis and epithelial (Fig. 4), and endometrial cells of the uterus (Fig. 5). [Conclusion] Our study has shown that exogenous gonadotropin used for superovulation can promote the expression of epidermal growth factor expression in ovaries, fallopian tubes and uterus, suggesting that EGF may be involved in the reproductive process of Hyla Rabbit and regulate the functions of ovaries, fallopian tubes and uterus.