Objective To establish an efficient method to isolate and culture rat bone marrow mesenchymal stem cells (BMSCs) and apply PKH26 to label them in vitro to explore the effect of PKH26 labeling on their biological characteristics and achieve in vitro tracing. Methods Hind limb bones of 5-day-old rats were separated, surrounding muscle and fascia were removed, and the bones were cut into small pieces for culture. BMSCs were purified by fluid exchange and passaging. Cell surface antigens of third passage cells were analyzed by flow cytometry. Under the same culture conditions, third passage BMSCs were labeled with PKH26. Cell morphology and proliferation of labeled and unlabeled groups were observed under a fluorescence microscope. Adipogenic induction characteristics and identification of labeled and unlabeled groups were compared. Results The bone marrow slice method was used to separate hind limb bones of 5-day-old mice. The BMSC shape was slender, spindle, and uniform. A large number of BMSCs was rapidly obtained in a short time. Flow cytometry showed CD29 expression in (91. 18± 1. 29) % of cells, CD90 expression in (95. 04 ± 0. 11)% of cells, and CD45 expression in (1. 74 ± 0. 36) % of cells. PHK26 labeling had no significant effect on the morphology or proliferation of BMSCs ( P> 0. 05) or induction of osteogenesis or adipogenesis. Conclusions A large number of high-purity BMSCs was rapidly cultured by the method from 5-day-old rat bone marrow slices, which can be used as seed cells for bone tissue engineering. PKH26 can also label rat BMSCs in vitro.