Technical Abstract: Autophagy is a pathway in eukaryotes by which nutrient remobilization occurs through bulk protein and organelle turnover. Autophagy not only aides cells in coping with harsh environments but also plays a key role in many physiological processes that include pollen germination and tube growth. Most autophagic components are conserved among eukaryotes, but phylum-specific molecular components also exist. We show here that Arabidopsis thaliana PTEN, a protein and lipid dual phosphatase homologous to animal PTENs (phosphatase and tensin homologs deleted on chromosome 10), regulates autophagy in pollen tubes by disrupting the dynamics of phosphatidylinositol 3-phosphate (PI3P). The pollen-specific PTEN bound PI3P in vitro and was localized at PI3P-positive vesicles. Overexpression of PTEN caused accumulation of autophagic bodies and resulted in gametophytic male sterility. Such an overexpression effect was dependent upon its lipid phosphatase activity and was inhibited by exogenous PI3P or by expression of a class III phosphatidylinositol 3-kinase (PI3K) that produced PI3P. Overexpression of PTEN disrupted the dynamics of autophagosomes and a subpopulation of endosomes, as shown by altered localization patterns of respective fluorescent markers. Treatment with wortmannin, an inhibitor of class III PI3K, mimicked the effects by PTEN overexpression, which implied a critical role for PI3P dynamics in these processes. Despite sharing evolutionarily conserved catalytic domains, plant PTENs contain regulatory sequences that are distinct from those of animal PTENs, which might underlie their differing membrane association and thereby function. Our results show that PTEN regulates autophagy through phylum-specific molecular mechanisms.