Objective To explore the feasibility of labeling in vitro rabbit osteoblasts with Qtracker and the features of Qtracker-labeled rabbit osteoblasts. Methods A healthy male rabbit, 3 months old, weighing 2 kg, was used in this study. After bone marrow was aspirated, bone marrow stromal cells (BMSCs) were isolated and cultured using the adherence method in vitro. The third passage of BMSCs was induced into osteablasts before incubation with Qtracker at concentrations of 1, 2, 4, 8, 16, 32 nmol/10~6 cells (Groups A, B, C, D, E, F respectively). Cells not labeled by Qtracker served as negative control (Group G). The following parameters were measured: induction, differentiation and determination of rabbit osteoblasts; the optimal mass concentration of Qtracker labeling by fluorescence microscopy and flow cytometry; the cell sur-vival rates at various concentrations of Qtraeker labeling by trypan-blue exclusion; Qtracker-labeled cell pro-liferation by MTr. Results The primary and the passage rabbit BMSCs were chiefly of fusiform shape. Rabbit BMSCs differentiated into osteoblasts following induction. The osteoblasts cytoplasm showed green fluorescence under fluorescence microscopy after being labeled by Qtracker. The mean labeling rate increased with the increased concentration of Qtracker, reaching up to (93.58±2.08) % after incubation at 8 nmol/ 10~6 cells by fluorescence microscopy, and (95.24±1.31) % by flow cytometry. There were no significant differences between Groups D, E, F(P>0.05), but significant differences were found between Groups A, B, C and Groups D, E, F (P<0.05). The labeling rate for Group G was 0. The cell survival rates were all above 96% (P>0.05) . No significant differences were found in the cell proliferation among various con-centrations (P>0.05). Conclusions Qtraeker can be used as a labeling marker for rabbit osteoblasts. When the concentration is at 8 nmol/10~6 cells, optimal labeling effect can be achieved. Rabbit osteoblasts labeled with Qtracker are of high efficiency and safety.