Objective To determine the effect of the supernatant of adipose-derived mesenchymal stem cells (AD-MSCs) on macrophages phenotype cultured under high glucose or combined with inflammation circumstance. Methods Rat peritoneal macrophages were isolated and then cultured under 33 mmol/L glucose,1 μg/ml LPS and their combination separately for 12 h, then the culture media were added with 0.5 ml supernatant of AD-MSCs. After 48h,the morphology of the macrophages with the supernatant intervention or not was observed with microscopy. The percentages of M1 and M2 macrophages were detected by flow cytometry, and the concentrations of cytokines IL-6, IL-10 and PGE2 were determined with ELISA.Results Compared with normal control cells, the percentage of M1 macrophage,M1/M2 ratio and IL-6 concentration were significantly higher, while the proportion of M2 macrophages and contents of IL-10 and PGE2 were obviously declined in the macrophages treated with high glucose, LPS or their combination. However, addition of the MSCs supernatant resulted in notable decrease in the percentage of M1 macrophages and M1/M2 ratio, and significant increases in the proportion of M2 macrophage in the LPS and combination groups. The results of ELISA indicated that MSCs supernatant reduced the level of IL-6 while elevated those of IL-10 and PGE2 in the macrophages treated with high glucose, LPS or their combination. Conclusion Combination of high glucose and LPS synergistically aggravates the polarization of pro-inflammatory M1 macrophages, and promotes the secretion of inflammatory cytokines. The supernatant of MSCs improves M2 phenotype polarization and production of anti-inflammatory cytokines.