〔Abstract〕 Objective To investigate the effect of co-culture of adipose-derived stem cells(ADSC) and endothelial progenitor cells(EPC) on the activity of EPC and its related mechanism. Methods Rat ADSC and EPC were isolated, cultured, expanded and identifiedin vitro. The experiment was divided into three groups: EPC group, EPC+ADSC co-culture group, and EPC+ADSC+PI3K-inhibitor group. After 48 hours of co-culture, the cells of the three groups were treated with Transwell. The effects of ADSC and EPC co-culture and PI3K/AKT pathway on EPC activity were evaluated by CCK-8 assay, scratch assay and angiogenesis assay, respectively. Western blot was used to detect vascular endothelial growth factor A(VEGFA) and endothelial nitric oxide synthase(eNOS),vascular endothelial-cadherin(VE-cadherin), CD133, phospho-phosphatidylinositol 3-kinase(phospho-phosphatidylinositol 3-kinase(p-PI3K) and phospho-protein Kinase B(p-AKT) expression levels in EPC to detect the effects of ADSC and EPC co-culture and PI3K/AKT pathway on the differentiation ability of EPC into mature endothelial cells. Results CCK-8 results showed that the absorbance at 450 nm of EPC in EPC+ADSC co-culture group was higher than that in EPC group and EPC+ADSC+PI3K-inhibitor group at different time points, and the difference was statistically significant(P<0.01). The scratch test showed that the relative scratch distance of EPC+ADSC co-culture group was smaller than that of EPC group and EPC+ADSC+PI3K-inhibitor group after 24 hours, and the difference was statistically significant(P<0.01). Tube formation assay showed that the average number of tube-like structures formed in EPC+ADSC co-culture group was higher than that in EPC group and EPC+ADSC+PI3K-inhibitor group, and the difference was statistically significant(P<0.01). Western blot showed that the expression levels of VEGFA, eNOS, VE-cadherin, p-PI3K and p-AKT of EPC in EPC+ADSC co-culture group were higher than those in EPC group and EPC+ADSC+PI3K-inhibitor group. The expression level of CD133 in EPC group was lower than that in EPC+ADSC+PI3K-inhibitor group, and the difference was statistically significant(P<0.01). Conclusion Co-culture of ADSC and EPC can improve the proliferation, migration, differentiation and vasogenic activity of EPC through the regulation of PI3K/AKT pathway.