This study was purposed to establish the methods for isolation, culture, identification and labeling of bone marrow mesenchymal stem cells (BMMSC), so as to provide quantified seed cells for cell transplantation. Bone marrow was collected from SD rat by flushing femur and tibias under sterile condition and BMMSC were purified by adherent culture and amplified in vitro. The immunophenotypes of BMMSC were identified by flow cytometry, the ability of differentiation to osteogenic and adipogenic lineages was detected by alizarin red and oil red O respectively. The BMMSC were transfected by using lentivirus with green fluorescence protein (GFP) gene so as to determine GFP expression in BMMSC. The results demonstrated that the method of adherent culture could effectively isolate and purify rat BMMSC which displayed homogenous fibro-like morphology. The flow cytometry showed that BMMSC expressed CD29, CD44, not expressed CD34, CD45. The BMMSC could differentiated into osteoblasts and adipocytes two mesenchymal lineages when grown in specific medium for each lineage. After being transfected by lentivirus, BMMSC could express GFP. It is concluded that the adherent culture is simple, effective, feasible method to separate MSC from the bone marrow of adult rats; the separated and cultured cells exhibit the biological characteristics of BMMSC and differentiating potential. BMMSC can express GFP efficiently and stably in vitro after being transfected by lentivirus, which can be used to label cells for tracing in vivo.