Our previous investigations identified miR-30a-3p as a differentially expressed miRNA in ovine mammary tissue across sheep breeds with distinct lactation performance and different physiological stages. However, its regulatory mechanisms controlling mammary gland development and lactation remain unexplored. In this study, the effect of miR-30a-3p on the proliferation of ovine mammary epithelial cells (MECs) and the target genes of miR-30a-3p were investigated. The regulatory effects of miR-30a-3p on the expression of the target genes and the content of triglycerides in ovine MECs were also analyzed. The transfection of miR-30a-3p mimic was found to promote cell viability and the number of proliferated ovine MECs using CCK8 and Edu assays. On the contrary, the miR-30a-3p inhibitor showed the opposite results with the miR-30a-3p mimic. These results suggest that miR-30a-3p promotes the proliferation of ovine MECs. The dual luciferase assay revealed that Phosphatase and Tensin Homolog () can be targeted with miR-30a-3p. The transfection of miR-30a-3p mimic into ovine MECs resulted in a significant decrease in expression levels of the target genes. However, it promotes five milk fat synthesis marker genes, , , , , and , and promotes the content of triglycerides. The opposite effect of miR-30a-3p inhibitor with mimic on the expression levels of these genes and the level of triglycerides was also observed. This is the first study to reveal the biological mechanisms by which miR-30a-3p promotes milk fat synthesis by targeting in sheep.