Bone marrow mesenchymal stem cells (BMSCs) are promising tool for cell therapy. However, their biological behaviors and functions could be altered by the surrounding microenvironment. In this study, we intend to investigate the effects of septic microenvironment on the biological behaviors of BMSCs. Sepsis was induced in healthy Sprague-Dawley rats through cecal ligation puncture procedure. BMSCs were isolated and expanded from these septic animals in vitro. BMSCs from sham control rats were cultured as control. Morphology of cells was detected by light microscope. Proliferation and self-renewal of BMSCs were detected through cell counting kit-8 and colony forming unit-fibroblasts test. Cell cycle and apoptosis were detected by flow cytometry. Cell migration was assessed through transwell assay. Senescence-associated molecules were analyzed through Western-blot. Capacity of differentiation and activity of senescence-associated β-galactosidase (β-gal) were detected as well. Our results showed that the ability of proliferation and migration was significantly inhibited in BMSCs from septic animals, and cell cycle was blocked in G1 phase with enhanced expression of P21 and P16. Furthermore, expression of sirtuin1 was decreased and activity of β-gal in these cells was significantly enhanced. These results indicated that septic microenvironment induced a senescence-associated phenotype in BMSCs.