Perturbations in the intracellular PtdIns 3,5-P₂ pool or the downstream transmission of PtdIns 3,5-P₂ signals often result in a gradual development of gross morphological changes in the pleiomorphic multivesicular endosomes, culminating with the appearance of cytoplasmic vacuoles. To identify the onset of PtdIns 3,5-P₂ functional requirements along the endocytic system, we characterized here morphological changes associated with early expression of the dominant-negative kinase-deficient form (K1831E) of the PtdIns 3,5-P₂-producing kinase PIKfyve, prior to formation of cytoplasmic vacuoles in transfected COS cells. Enlarged PIKfyve^K1831E-positive vesicles colocalizing with dilated EEA1- and Rab5a^WT-positive perinuclear endosomes were observed. This was dependent on the presence of active forms of Rab5 and the generation of PtdIns 3-P-enriched platforms on early endosomes. Because PIKfyve^WT did not substantially colocalize with EEA1- or Rab5-positive endosomes in COS cells, the dynamic PIKfyve-catalyzed PtdIns 3-to-PtdIns 3,5-P₂ switch was suggested to drive away PIKfyve^WT from early endosomes toward later compartments. Late endosomes/lysosomes marked by ectopically expressed LAMP1 or Rab7 were dislocated from their typical perinuclear position upon PIKfyve^K1831E early expression. Cytosols derived from cells stably expressing PIKfyve^K1831E stimulated endosome fusion in vitro, whereas PIKfyve^WT-enriched cytosols had the opposite effect, consistent with PtdIns 3,5-P₂ production negatively regulating the endosome fusion. Together, our data indicate that PtdIns 3,5-P₂ defines specific endosome platforms at the onset of the degradation pathway to regulate the complex process of membrane remodeling and dynamics.