Microtubule‐associated protein 1 light chain 3 (LC3) is a unique modifier protein. LC3‐I, the cytosolic form, is modified to LC3‐II, the membrane‐bound form, by a mechanism similar to ubiquitylation by E1‐ and E2‐like enzymes, Apg7p and Apg3p, respectively. In the present study, we found that LC3‐I is processed to LC3‐II during the differentiation and recovery from puromycin aminonucleoside‐induced nephrosis of podocytes. LC3 is especially expressed in the podocytes of rat kidney as the membrane‐bound form LC3‐II. Biochemical analysis using a conditionally immortalized mouse podocyte clone (MPC) revealed that LC3‐I is processed to LC3‐II during the differentiation of cells into mature podocytes and accumulates in the membrane‐rich fraction of the cell lysate. LC3‐II‐localized vesicles, which differ from lysosomes and endosomes, in differentiated MPC cells are morphologically similar to autophagic vacuoles during starvation‐induced autophagy. During starvation‐induced autophagy, autophagosomes fuses with lysosome and LC3‐II on autophagosomes is finally degraded by lysosomal proteases. However, in differentiated MPC cells, little LC3‐II on the vesicles is degraded by lysosomal proteases, suggesting that little LC3‐II‐localized vesicles in differentiated MPC cells fuse with lysosome. Furthermore, the LC3‐II level in differentiated MPC cells increases with recovery from damage caused by experimental puromycin aminonucleoside‐induced nephrosis. These results suggest that LC3‐II‐localized vesicles play an important role in the physiological function of podocytes.