Three Luxi adult Yellow steers were used to isolate and culture intramuscular pre-adipocytes in vitro as well as to examine factors influencing their proliferation and differentiation. The intramuscular pre-adipocytes were taken from adipose tissues within muscles between the sixth and seventh rib and cultured after digestion with collagenase I. The results showed that the separated cell populations were highly homogeneous, proliferative and doubled within 62 h. When the confluent pre-adipocytes were treated with 10 μg/ml insulin and 0.25 μmol/l dexamethasone, small lipid droplets appeared on day 2 and the number of lipid droplets rapidly increased around the nuclei on day 6. Their dynamic morphological changes, growth curve, Oil Red O staining, and reaction to insulin and dexamethasone all verified their pre-adipocyte identity. Under controlled conditions, the intramuscular pre-adipocytes resumed proliferating and differentiating in vitro. Interestingly, the proportion of cultured diploid pre-adipocytes reached more than 90% after six repeated cultures. This study confirms the existence of functionally active pre-adipocytes within the muscles of Chinese adult local breed cattle. These cell strains are a potentially useful model for understanding further the mechanism of intramuscular adipose deposition in tissues, in order to improve beef quality based on Chinese local breed beef cattle.