The purpose of the present study was to investigate the regulation of propionate on the proliferation and differentiation of cultured intramuscular preadipocytes from three adult Luxi Yellow steers. The flow cytometry, lipid droplets stained by oil red O, and cytoplasmic fat content were detected after cultured cells were treated with different concentrations of propionate (0, 1, 3, 6 mM). The results showed that the separated cells were highly homogeneous and proliferative. Propionate (1 mM) did not significantly affect (P>0.05) the cell proportion of phase G0/G1, but remarkably (P<0.05) increased the proportion of phase S+G2/M. However, the cell proportion significantly increased (P<0.05) in the phase G0/G1, and decreased (P<0.05) in the phase S+G2/M when the concentration of propionate reached to 6 mM. When the confluent intramuscular preadipocytes were treated with insulin (10 µg/ml) and dexamethasone (0.25 µM), small lipid droplets began to appear, and their number rapidly increased around the nuclei on day 6. Subsequently, when the confluent intramuscular preadipocytes were treated with propionate, significant accumulation of lipid droplets was observed on day 6, and lipid accumulation extent was dependent on the dose of propionate. In addition, cytoplasmic fat contents of cultured preadipocytes treated with propionate (1, 3, 6 mM) for 6 days were 1.5, 2, and 2 times higher than those of control group (without propionate addition). In conclusion, the present study confirmed that there were indeed functionally active preadipocytes existing in the muscle of Chinese adult Luxi steers. Propionate may play an important physiological role in regulation of adipogenesis and stimulation of proliferation and differentiation of Luxi bovine intramuscular preadipocytes.