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Background Intramuscular fat (IMF) content is the major evaluating indicator in chicken meat quality. We previously found that miR-128-3p was differentially expressed in chicken intramuscular adipocyte at different periods. However, little is known about miR-128-3p associated with intramuscular adipocyte differentiation. The purpose of this present study was to investigate the biological functions and regulatory mechanism of miR-128-3p in chicken intramuscular adipogenesis. Results A series of assay results confirmed that miR-128-3p could promote the proliferation of intramuscular adipocytes and inhibit cell differentiation. A total of 223 and 1,050 differentially expressed genes (DEGs) were identified in the mimic treatment group and inhibitor treatment group compared with the control group, respectively. Functional enrichment analysis revealed that DEGs involved in lipid metabolism related processes, such as MAPK signaling pathway and TGF-β signaling pathway. Furthermore, target genes prediction analysis showed that miR-128-3p can target multiple genes from DEGs, such as FDPS, GGT5, TMEM37, and ASL2. Luciferase assay showed that the miR-128-3p targeted to the 3’ UTR of FDPS. Subsequent functional assays demonstrated that miR-128-3p acted as an inhibitor of intramuscular adipocyte differentiation by targeting FDPS. Conclusion This study found that miR-128-3p inhibited chicken intramuscular adipocytes differentiation by down regulating FDPS. Our findings provided a theoretical basis for the study of lipid metabolism, and also provided a potential target for molecular breeding to improve meat quality.