Bovine bone marrow-derived mesenchymal stem cells (bBMSCs) were isolated from the bone marrow of a 4-6-month-old fetal bovine and then characterized by immunofluorescence and reverse transcriptase polymerase chain reaction. We found that primary bBMSCs could be expanded for 46 passages; the total culture time in vitro was 125 days. The results of surface antigen detection showed that bBMSCs expressed CD29, CD44, and CD73 but did not express endothelial cells and hematopoietic cells-specific marker CD31, CD34, and CD45. The cells from four passages (passages 3, 9, 15, and 25) were successfully induced to differentiate into osteoblasts, adipocytes, hepatic, and islet-like cells. The results indicate the potential for multi-lineage differentiation of bBMSCs that may represent an ideal candidate for cellular transplantation therapy.