<p>The yak, a livestock breed native to Qinghai Province in China. However, the low intramuscular fat (IMF) content of yak meat negatively impacts its taste and flavor, reducing its relative economic value. The experiment was conducted by inhibiting the expression of miR-127 and miR-375 in yak precursor adipocytes. To explore its effect on the proliferation and differentiation of yak adipocyte precursors, and then analyze the molecular regulatory mechanisms of miR-127 and miR-375 on IMF deposition in yak. Furthermore, using RNA-Seq analysis, this study identified the differentially expressed genes (DEGs) and functional pathways regulated by miR-127 and miR-375 that influence IMF deposition. Cellular functional validation experiments showed that inhibiting miR-127 can suppress the proliferation of yak intramuscular preadipocytes and promote their differentiation. Meanwhile, the inhibition of miR-375 had the opposite effect, promoting proliferation and inhibiting differentiation. RNA-Seq analysis revealed that miR-127 inhibition altered the expression of several DEGs, including <i>IFNB1</i>, <i>IFNA2</i>, <i>FABP4</i>, <i>CCL5</i>, <i>ITPKA</i>, and <i>IP6K3</i>. Similarly, the inhibition of miR-375 affected the expression of genes such as <i>IFNB1</i>, <i>IFNA2</i>, <i>FABP4</i>, <i>CCL5</i>, <i>FOS</i>, and <i>IL6</i>. Functional enrichment analysis revealed that many of these DEGs were involved in key signaling pathways, such as the Phosphatidylinositol 3-Kinase-Protein Kinase B (PI3K-Akt) signaling pathway, the Mitogen-Activated Protein Kinase (MAPK) signaling pathway, and the Toll-like receptor (TLR) signaling pathway. Of these, the TLR signaling pathway showed the most significant enrichment of DEGs. Notably, <i>IFNB1</i>, <i>IFNA2</i>, and <i>IL6</i> appeared to regulate the proliferation and differentiation of intramuscular preadipocytes in yaks by activating the TLR signaling pathway, thereby influencing IMF deposition.</p>