【Abstract】 Objective To explore the optimal dosage, timing and cytotoxicity of bromodeoxyuridine (BrdU) labelling for rabbit adipose-derived stromal stem cells (ADSCs) in vitro so as to confirm its feasibil ity for stem cells labell ing and tracer means. Methods Six rabbits were used in this experiment, aged 8-12 weeks, weighing 1.5-2.0 kg and neglecting their gender. 1-2 mL fat was removed, the ADSCs were isolated and cultured using the adherence method in vitro . The 3rd passage of ADSCs was incubated with BrdU at 5, 10, 15 and 20 μg/mL (groups A, B, C and D）for 12, 24, 48 and 72 hours to identify the optimal BrdU concentration and incubating time for cell labell ing. Immunohistochemistry and trypanblau strain were performed respectively to calculate the labell ing index (positive rate) and the cells’ activity for different time after BrdU labell ing. The ADSCs without BrdU labell ing were used as control (Group E）. Results The main appearance of primary ADSCs was short fusiform shape, and of the 3rd passage ADSCs long fusiform shape. The 3rd passage of ADSCs could differentiate into osteoblastsand adipocytes under corresponding inductive medium. The ADSCs’ nucleus show green fluor under fluorescence microscope after labeled by the BrdU. The labell ing ratio increased in groups A, B, C and D after incubating 12 hours, the mean labell ing ratio were 30.6％ ±2.3％，32.4％ ±1.9％，45.8％ ±1.8％，50.8％ ±3.1％ , respectively, and the labell ing ratio of Group E was 0. There were significant differences between groups C, D and Group A (P lt; 0.01). The labell ing ratio of groups A, B, C and D were 45.9％ ±2.0％，87.9％ ±3.3％，90.6％ ±2.9％，91.7％ ±3.2％，respectively after 24 hours and the labell ing ratio of Group E was 0. There were significant differences between groups B, C, D and Group A (P lt; 0.01). The results of all groups after incubating48 hours and 72 h ours were similar to that after incubating 24 hours. The cell counting of groups A, B, C and D were better than that of Group E, but showing no siginificant differences（P gt; 0.05）. Conclusion The most appropriate time for BrdU labell ing ADSCs is 48 hours, the most appropriate concentration is 10 μg/mL. The labell ing rate is high and cytotoxicity is l ittle.