Abstract: Objective To study the optimal dosage and timing for bromodeoxyuridine(BrdU) labeling of rat bone marrow-derived mesenchymal stem cells(MSCs) in vitro. Methods Bone marrow-derived MSCs of SD rats were cultured in vitro routinely and the sixth passage was taken for identification of specific surface antigens by flow cytometry. Before reaching cell confluence, the purified MSCs were incubated with BrdU at different concentrations(5, 10, and 15 μmol/L) for different incubating time(3, 12, 24, 36, 48, and 72 h) with 10 μmol/L BrdU, to identify the optimal BrdU concentration and incubating time for cell labeling. Immunohistochemistry was performed to calculate the labeling index(LI). Result Flow cytometry showed that MSCs expressed CD29 and CD44 but not CD11b or CD45. Incubation of the MSCs with BrdU at 10 μmol/L and for an optimal length of 48 h appeared to achieve the highest LI, both of which exceeded 98%, with the labeling identifiable in five consecutive passages. Conclusions The continually passaged cells are MSCs, the incubation of which with 10 μmol/L BrdU for 48 h may achieve a LI over 98% without producing obvious cell damages. The results suggest that BrdU labeling provides a feasible means for a dynamic in vivo observation of the survival, growth and differentiation of the implanted MSCs.