Abstract: Smooth muscle progenitor cell (SPC) is a novel cell source with the capability of proliferating, migrating, and differentiating into smooth muscle cell. In our previous study, SPC had been successfully isolated from the peripheral blood of New Zealand Rabbit and demonstrated the potential of being used as a smooth muscle cell source for tissue engineering of urinary bladder. However, it is important and necessary to investigate whether SPCs can survive, proliferate, migrate, differentiate into smooth muscle cells and integrate into the host tissue after they are seeded into a scaffold and implanted for bladder augmentation. Cell tracer technique may help resolve this problem, which can be used to trace the cell fate after transplantation. It is important to select an appropriate cell tracer with a high labeling efficiency but without the side effects on cells. CM-DiI is such a fluorescent dye that has being used widely for cell labeling in tissue engineering. Therefore, the objective of this study was to evaluate the feasibility of using CM-DiI as a tracer to label SPC cultured from rabbit peripheral blood. In this study, SPCs were isolated and cultured from the peripheral blood of New Zealand Rabbit and labeled with CM-DiI according to the manufacturer’s instructions. Labeling rate was evaluated by fluorescence microscope and flow cytometry study. After cultured on coverslips, SPCs were detected by indirect immunofluorescent staining using monoclonal antibody against alpha-smooth muscle actin (α-SMA), Calponin and Proliferating Cell Nuclear Antigen (PCNA). The impact of CM-DiI on the growth of SPC was examined by trypan blue exclusion, cell adhesion, cell proliferation and cell migration assays. Our results demonstrated that SPCs emerged after four days of culture and could formed clones after one week in culture. The labeling rate using CM-DiI was about 96% as detected with fluorescence microscope and flow cytometry study. After continuous passage culture for two weeks, the labeling rate was still greater than 40%. In indirect immunofluorescent staining, SPCs showed positive staining for α-SMA, Calponin and PCNA after labeling. There were no significant differences in cell survival, adhesion, proliferation or migration between labeled and unlabeled SPCs. In conclusion, CM-DiI labeling didn’t change the phenotypes of SPCs. No side effects on the capabilities of SPCs adhesion, proliferation or migration were noted. Our results support the use of CM-DiI as an effective and safe cell tracer in SPC-based tissue engineering of bladder.