We recently found that both annexin A7 and integrin β4 were involved in autophagy of vascular endothelial cells. But, their relation is not clear. In this study, we addressed this question by using a small molecule ABO that promoted autophagy by targeting annexin A7. The results showed that knockdown of integrin β4 partly inhibited ABO-induced autophagy in vascular endothelial cells. Furthermore, in HEK293 cells that express integrin β4 too low to detect by western blot, ABO could not induce autophagy. If integrin β4 was overexpressed in HEK293 cells, ABO could evoke autophagy. On the other hand, knockdown of annexin A7 also blocked ABO-induced autophagy although the level of integrin β4 was elevated. Moreover, by co-immunoprecipitation, we identified the interaction of integrin β4 and annexin A7, and found that ABO could modulate the interaction, at the same time, the phosphorylation of Y-1494 in integrin β4 cytoplasmic domain was inhibited significantly in vitro and in vivo. Hence, by identifying the interaction between integrin β4 and annexin A7, we demonstrated that both annexin A7 and integrin β4 were essential for small molecule ABO-induced autophagy and targeting annexin A7 by ABO could modulate integrin β4 phosphorylation, while Y-1494 phosphorylation of integrin β4 may negatively regulate autophagy.