Intramuscular fat (IMF) plays a crucial role in enhancing the tenderness, flavor, and juiciness of beef, making the increase of IMF content a significant objective in beef breeding. A key factor influencing IMF levels is the number of intramuscular preadipocytes. Previous studies have indicated a correlation between bta-miR-484 and IMF content. In this study, we found that bta-miR-484 is differentially expressed during the proliferation of intramuscular preadipocytes. Our research identified that bta-miR-484 targets MAP3K9, revealing a novel mechanism for regulating both proliferation and apoptosis via the JNK signaling pathway. Functional gain and loss experiments demonstrated that bta-miR-484 inhibits the transition of bovine intramuscular preadipocytes from the G0/G1 phase to the S phase, and significant increase the proportion of early apoptotic cells. Additionally, miRNA pulldown and luciferase reporter assays confirmed MAP3K9 as the target gene of bta-miR-484. Furthermore, rescue experiments indicated that bta-miR-484 mediates its effects on proliferation and apoptosis through the MAP3K9/JNK/CCND1 and MAP3K9/JNK/BCL2 axes. These findings suggest that bta-miR-484 is a non-coding RNA that inhibits the proliferation and promotes the apoptosis of intramuscular preadipocytes, indicating that treatment with bta-miR-484 may offers a novel strategy for enhancing IMF content.