Abstract: Objective：To investigate the possible effects of cell membrane green fluorescent probe 3,3′ dioctadecyloxacarbocyanine perchlorate (DiO) on the biological function of human bone marrow mesenchymal stem cells (hBMMSCs) after labeling hBMMSCs in vitro. Methods：hBMMSCs were separated by Ficoll density gradient centrifugation, and then labeled by DiO. The labeling efficiency of DiO was detected by flow cytometry. The effects of DiO on the migration, proliferation and multi directional differentiation potential of hBMMSCs were analyzed by Transwell migration assay, colony formation assay, growth curve analysis, and adipogenic and osteogenic induction assay, respectively. Results：The efficiency of DiO labeling hBMMSCs reached to (99.5±0.4)%. There were no significant differences in the migration, proliferation, and adipogenic and osteogenic differentiation potential between DiO labeled hBMMSCs and unlabeled hBMMSCs (P＞0.05). Conclusion：DiO labeling hBMMSCs does not affect their biological function, indicating that DiO may be a safe and effective green fluorescent dye for labeling hBMMSCs in vitro.