Beef quality is critically determined by intramuscular fat (IMF) deposition. Retinoic acid (RA), the active metabolite of vitamin A, plays an essential regulatory role in IMF development. To systematically investigate RA-mediated regulation of IMF formation in cattle, we established a concentration gradient of RA supplementation and employed a systematic screening approach to identify the optimal dosage for modulating bovine intramuscular adipocytes (IMAs) proliferation and differentiation. Subsequently, leveraging integrated multi-omics approaches, we screened the key downstream molecular targets through which RA governs IMF biosynthesis, and clarified the potential regulatory mechanism of this target. Our experimental data establish that RA promotes the proliferation of IMAs through modulation of G1/S phase progression. Concurrently, RA enhances triglyceride biosynthesis in IMAs by activating PPARγ-mediated cell differentiation and LPL-mediated intracellular lipid accumulation. Integrated transcriptomics and metabonomics analyses identified FABP4, CD36, EBF2, LRP1 and CAV1 as key candidate genes involved in RA-mediated IMF production. Functional interrogation revealed that FABP4 knockdown markedly attenuated lipid accumulation in IMAs, a phenotype rescued through RA supplementation, confirming FABP4 as the critical effector mediating vitamin A's regulation of bovine IMF deposition. These results provide a new understanding of how nutritional factors affect beef quality at the molecular level.