Immature Sertoli cell proliferation determines the final number of mature Sertoli cells and further regulates spermatogenesis. Accumulating evidence demonstrated that microRNAs (miRNA) play an important role in Sertoli cell proliferation, differentiation, and apoptosis. However, the effect and molecular mechanism of miRNA on bovine immature Sertoli cells remains to be poorly understood. In this study, miRNA sequencing of testes collected in mature (24-month-old) and immature (neonatal) bulls was conducted to determine the miRNA expression profiles. MicroRNA-34b was one of the differentially expressed miRNA and was selected for in-depth functional studies pertaining to Sertoli cell growth. The results showed that miR-34b mimic transfection in primary Sertoli cells (PSC) inhibited cell proliferation and induced cell cycle arrested at G2 phase and decreased the expression of cell cycle-related genes CCNB1, CDK1, CDC25C, and C-MYC. MicroRNA-34b overexpression also lead to increased cell apoptosis, with pro-apoptotic genes P53 and BAX upregulated, while anti-apoptotic gene BCL2 decreased. However, miR-34b knockdown had the opposite effects. Through a combination of transcriptome sequencing, bioinformatics analysis, dual luciferase reporter assay, and western blotting, mitogen-activated protein kinase kinase1 (MAP2K1), also known as MEK1, was identified as a target of miR-34b. In addition, PSC proliferation inhibition was mediated by cell cycle arrest and apoptosis with MAP2K1 interference. Overexpression of MAP2K1 effectively reversed the miR-34b-repressed PSC cell growth. Moreover, both miR-34b overexpression and MAP2K1 knock-down decreased the protein levels of P-ERK1/2, while MAP2K1 overexpression showed opposite effects. In summary, data suggest that miR-34b regulates PSC proliferation and apoptosis through MEK/ERK signaling pathway. These data provide a theoretical and experimental framework for further clarifying the regulation of cell growth in PSC of bovine.