Objective To optimize the in vitro culture system of C57/BL6 marrow mesenchymal stem cells (MSCs) and to investigate the effect of alcohol and acetaldehyde on MSCs. Methods The MSCs were isolated from the femur marrow of C57/BL6 mice and were cultured in the optimized system, so that highlypurified MSCs were harvested and identified by immunohistochemistry. Then, MSCs were cultured in the medium containing alcohol or its metabolic product acetaldehyde, with the following concentration groups: alcohol 5.7，17.0，50.0，100.0 and 150.0 mmol/L; acetaldehyde 4.5, 0.9, 0.18, 0.036, 0.007 2, 0.001 44 , 0.000 28 mmol/L. MSCs were cultured with α-MEM as the control group. After 3 days, their proliferation activity was measured by the MTT method. Results MSCs within 6 passages had a good stability and a high proliferation activity. They were identified to express CD90 but no CD34. The MTT assay showed that alcohol at the concentration greater than 100.0 mmol/L and acetaldehyde at the concentration greater than 4.5 mmol/L could inhibit proliferation of MSCs(P＜0.05) . But the proliferation activity might rise with an increase in the acetaldehyde concentration smaller than 0.18 mmol/L(P＜0.05) . Conclusion Theoptimized culture system can effectively isolate and culture MSCs. Both alcoholand acetaldehyde can inhibit proliferation of MSCs but toxicity of acetaldehydeis more serious.