Prolactin (PRL) has been reported to influence reproductive performance and cell apoptosis. However, its mechanism remains unclear. Hence, in the present study, ovine ovarian granulosa cells (GCs) were used as a cell model to investigate the relationship between PRL concentration and GC apoptosis, as well as its possible mechanisms. We examined the relationship between serum PRL concentration and follicle counts in sexually mature ewes. GCs were isolated from adult ewes and treated with different concentrations of PRL, while 500 ng/mL PRL was selected as the high concentration of prolactin (HPC). Then, we applied the transcriptome sequencing (RNA-Seq) combined with a gene editing approach to explore the HPC contributing to cell apoptosis and steroid hormones. The apoptosis of GCs gradually increased at PRL concentrations above 20 ng/mL, while 500 ng/mL PRL significantly decreased the secretion of steroid hormones and the expression ofL-PRLRandS-PRLR. The results indicated that PRL regulates GC development and steroid hormones mainly through the target geneMAPK12. The expression ofMAPK12was increased after knocked-downL-PRLRandS-PRLR, while it decreased after overexpressedL-PRLRandS-PRLR. Cell apoptosis was inhibited and the secretion of steroid hormones increased after interfering withMAPK12, while the overexpression ofMAPK12showed the opposite trend. Overall, the number of follicles gradually decreased with increasing PRL concentration. HPCs promoted apoptosis and inhibited steroid hormone secretion in GCs by upregulatingMAPK12through reducingL-PRLRandS-PRLR.