Objective To explore the homing of DiR labeled regulatory T cells ( Tregs) in human-ized heterologous liver tissue transplantation mouse model. Methods The fluorescence intensities of Tregs labeled with different concentrations of DiR dye and different incubation times were measured, and the cell viability was measured by 3-( 4, 5-dimethylthiazol-2-yl )-5-( 3-carboxymethoxyphenyl )-2-( 4-sulfophenyl )-2H-tetrazolium ( MTS) assay to determine the optimal incubation time and dye concentration. The effect of DiR dye on the function and phenotype of Tregs was verified by flow cytometry. The xenogeneic liver tissue transplantation mouse model was constructed and the immune system was reconstituted. Small animal fluores-cence imaging was performed at different time points after infusion of Tregs. Immunohistochemistry analysis was used to analyze immune reconstitution and lymphocyte distribution in vivo. One-way analysis of variance and Dunnett-t test were used to analyze the data. Results With the increase of DiR concentration and incu-bation time, the fluorescence intensity of Tregs increased and gradually weakened after reaching the peak at 3 d. The cell viability of the 5. 00 μg/ml and 20. 00 μg/ml groups was significantly lower than that of the control group (culture medium) at various time points (F=120.142-182.025, t=9.969-19.329, all P<0. 05) . After incubation for 30 min and 60 min, the activity of Tregs was also significantly lower than that of the control group (F=21.826-301.968, t=6.897-40.016, all P<0.05). Tregs were finally co-incubated with DiR dye at a concentration of 2.50 μg/ml for 5 min, which was used further in vivo experiments. The flow cytometry showed that DiR dye did not affect the phenotype or the function of Tregs. The small animal fluorescence imaging showed that Tregs could locate in the graft area of mouse model. Immunohistochemical analysis showed that Tregs could improve lymphocyte infiltration induced by immune reconstitution. Conclu-sion After labeling Tregs with DiR dye, the distribution of Tregs can be directly observed by fluorescence imaging, which is a promising imaging method for Tregs tracer.