Prolactin (PRL) is a pituitary hormone that regulates multiple physiological processes. However, the mechanisms of PLR synthesis have not been fully elucidated. The aims of the present study were to study the functions and the related mechanisms of miR-375 regulating PRL synthesis. We initially found that miR-375 mainly expressed in the lactotrophs of mouse pituitary gland. To identify the function of miR-375 in the pituitary gland, the miR-375 knockout mice were generated by using Crispr/Cas9 technique. The results showed that miR-375 knockout resulted in the decline of pituitary PRL mRNA and protein levels by 75.7% and 60.4% respectively, and the serum PRL level reduced about 46.1%, but had no significant effect on FSH, LH and TSH. Further, we identified that Estrogen receptor 1 (alpha) (Esr1) was a downstream molecule of miR-375. The real-time PCR and western blot results showed that ESR1 mRNA and protein levels markedly decreased by 40.9% and 42.9% in the miR-375 knockout mouse pituitary, and these were subsequently confirmed by the in vitro study using transfections of miR-375 mimics and inhibitors in pituitary lactotroph GH4 cells. Further, Rasd1 was predicted by bioinformatic tools and proved to be the direct target of miR-375 in lactotrophs using dual-luciferase reporter assay. Rasd1-siRNA transfection results revealed the negative effect of Rasd1 in regulating ESR1. Collectively, the results presented here demonstrate that miR-375 positively modulates PRL synthesis through Rasd1 and Esr1, which are crucial for understanding the regulating mechanisms of pituitary hormone synthesis.