Objective:To explore the relationship between the changes of autophagy and the number of mitochondria in the process of the differentiation of 3T3-L1 cells. Methods:(1)3T3-L1 preadipocytes were cultured and then induced by media mixed with insulin,DEX,IBMX. Lipid droplets were observed on the fifth day. The peroxisome proliferation activated receptor γ(PPARγ)and CAAT Enhancer Binding Proteins α(CEBPα)protein were detected by Western blotting. (2)Autophagosome pictures were taken by transmission electron microscope (TEM)and LC3II level was detected by Western blotting. Results:The TEM showed that during the differentiation of 3T3-L1 preadipocyte,autophagy vesicles appeared on the second day,extended to greater range of cytoplasm on the fourth day,then gradually disappeared from the sixth day. While the expression of LC3Ⅱ began to increase in the second day, reached the highest on the fourth day,and then gradually declined. Furthermore,with the change of autophagy vesicles during the differentiation,the number of mitochondria increased after a decrease. Conclusion:There are a lot of autophagy vesicles in the early and middle stage of the differentiation of 3T3-L1,including some mitophagy,which may be one of the reasons for the decrease of the number of mitochondria in early stage of differentiation.