MicroRNAs (miRNAs) are short non-coding RNA that post-transcriptionally regulates gene expression. miRNA-143 has been proposed to play a role in the differentiation of preadipocytes. However, effects and mechanism of miRNA-143 in the differentiation of mammals intramuscular adipocytes is unknown. In this study, the fibroblast-like preadipocytes were cultured from the marbling muscle tissue of holstein steers by the ceiling culture method. The in vitro studies showed that the fibroblast-like preadipocytes could differentiated into mature adipocytes with up-regulated expression of miRNA-143. Furthermore, the transfection of the fibroblast-like preadipocytes with miRNA-143 antisense inhibitor induced a significant suppression of differentiation, and indicated by decreased storage of lipid droplets and down-regulated expression of key adipocytes regulatory genes such as CCAAT/enhancer-binding protein-α (C/EBPα) and fatty acid binding proteins-4 (FABP-4). On the contrary, cells proliferation were increased with miRNA-143 inhibitor transfection. Taken together, our study provide the first evidence for stimulation of endogenous miRNA-143 in the differentiation of bovine intramuscular fat, which in part contribute to the regulated expression of adipocyte genes.