Prolactin (PRL) plays an important role in animal follicle development and ovulation. However, its regulatory effects on the different stages of the estrus cycle in ewes are unclear. In this study, bromocriptine (BCR, PRL inhibitor) was used to study the effect of PRL on the secretion of reproductive hormones and gene expressions in order to explore its regulatory effects on the sexual cycle of ewes. Eighty healthy ewes with the same parity and similar weights were randomly assigned to the control group (C, n = 40) and the treatment group (T, n = 40, fed bromocriptine). After estrus synchronization, thirty-one ewes with overt signs of estrus were selected from each group. Six blood samples were randomly obtained by jugular venipuncture to measure the concentration of PRL, estrogen (E2), progesterone (P4), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and gonadotropin-releasing hormone (GnRH) in the proestrus, estrus, metestrus, and diestrus. At the same time, we collected the ovaries of the six ewes in vivo after anesthesia in order to detect follicle and corpus luteum (CL) counts and measure the expression of hormone-receptor and apoptosis-related genes. The results show that PRL inhibition had no significant effects on the length of the estrus cycle (p > 0.05). In proestrus, the number of large and small follicles, the levels of E2, FSH, and GnRH, and the expressions of ER, FSHR, and the apoptotic gene Caspase-3 were increased (p < 0.05); and the number of middle follicles and the expression of anti-apoptotic gene Bcl-2 were decreased (p < 0.05) in the T group. In estrus, the number of large follicles, the levels of E2 and GnRH, and the expressions of the StAR, CYP19A1, and Bcl-2 genes were increased (p < 0.05), and the number of middle follicles was decreased (p < 0.05) in the T group. In metestrus, the number of small follicles and the expression of LHR (p < 0.05) and the pro-apoptotic gene Bax were increased (p < 0.05); the number of middle follicles was decreased (p < 0.05) in the T group. In diestrus, the number of large follicles, middle follicles, and CL, the level of P4, and the expressions of PR, 3β-HSD, StAR, Caspase-3, and Bax were increased (p < 0.05); the number of small follicles and the expression of Bcl-2 were decreased (p < 0.05) in the T group. In summary, PRL inhibition can affect the secretion of reproductive hormones, the follicle count, and the gene expression during the estrus cycle. These results provide a basis for understanding the mechanisms underlying the regulation of the ewe estrus cycle by PRL.