Recent studies have suggested that autophagy is a key mechanism in maintaining the integrity of podocytes. The mammalian homologue of yeast vacuolar protein sorting defective 34 (mVps34) has been implicated in the regulation of autophagy, but its role in podocytes is unknown. We generated a line of podocyte-specific mVps34-knockout (mVps34(pdKO)) mice, which were born at Mendelian ratios. These mice appeared grossly normal at 2 weeks of age but exhibited growth retardation and were significantly smaller than control mice by 6 weeks of age, with no difference in ratios of kidney to body weight. mVps34(pdKO) mice developed significant proteinuria by 3 weeks of age, developed severe kidney lesions by 5-6 weeks of age, and died before 9 weeks of age. There was striking podocyte vacuolization and proteinaceous casts, with marked glomerulosclerosis and interstitial fibrosis by 6 weeks of age. Electron microscopy revealed numerous enlarged vacuoles and increased autophagosomes in the podocytes, with complete foot process effacement and irregular and thickened glomerular basement membranes. Immunoblotting of isolated glomerular lysates revealed markedly elevated markers specific for lysosomes (LAMP1 and LAMP2) and autophagosomes (LC3-II/I). Immunofluorescence staining confirmed that the enlarged vacuoles originated from lysosomes. In conclusion, these results demonstrate an indispensable role for mVps34 in the trafficking of intracellular vesicles to protect the normal cellular metabolism, structure, and function of podocytes.