Objective To study the effect of Transwell co-culture of rat BMSCs and ADSCs on the osteogenic differentiation of ADSCs in vitro. Methods ADSCs and BMSCs were obtained from the same 6-week-old SD male rats. The second passage of ADSCs and BMSCs were harvested and placed into Transwell co-culture system at a ratio of 1 ∶1(co-culture group), ADSCs were placed in the bottom and BMSCs in the upper. Meanwhile ADSCs group and BMSCs group were served as control group. After 21 days of osteogenic induction and differentiation, the qualitative and semi-quantitative staining analysis of alizarin red were used to investigate the effect of BMSCs on ADSCs osteogenic differentiation. Results The results of semi-quantitative determination of alizarin red in the co-culture group were statistically different from that in the ADSCs group(P0.05). Conclusion Osteogenic mineralization ability was enhanced after in vitro Transwell co-culture of BMSCs and ADSCs, and BMSCs could promote osteogenic differentiation of ADSCs by paracrine.