Objective To establish a method for isolation, purification of bone marrow mesenchymal stem cells (MSCs) and for their differentiation into vascular endothelial cells, and to observe the regulatory role of microRNA126 expression during the differentiation. Methods MSCs were isolated from normal rat bone marrow using gradient density centrifugation and repeated attachment method. CD34, CD105 and CD73 expressions in MSCs were detected with immunofluorescent staining. MSCs were cultured with MEF medium for differentiating into endothelial cells; CD34, VE-cadherin and microRNA126 expressions were examined by qRT-PCR at different time points. Results Immunofluorescent detection demonstrated that MSCs have been isolated and purified successfully, with MSCs negative for CD34, strongly positive for CD105 and positive for CD73. The purified MSCs had a good uniformity and a purity above 90%. qRT-PCR examination revealed that CD34 and VE-cadherin expressions were not detected on day 1-4 of induction, strongly positive on day 5-6, and on day 7-9, VE-cadherin was still positive, CD34 decreased on day 7, and increased again on day 8-9. Interestingly, the expression of CD34 and microRNA126 mRNA was consistent during the differentiation. Conclusion We have successfully established a method for MSCs isolation and differentiation into endothelial cells; microRNA126 may play a regulatory role in MSCs differentiation into endothelial cells.