Dysregulation of the lipid kinase PIKfyve has been linked to neurodegeneration in mice and humans (Charcot Marie Tooth). We found that directly inhibiting PIKfyve activity in primary hippocampal neurons promotes an apoptosis-independent cell death. We have explored the mechanism underpinning this novel neurodegenerative pathway involving autophagic vesicular trafficking. While cell death was preceded by vacuolation of endolysosomal membranes there was no detectable difference in endocytosis of transferrin and WGA or in the retrograde trafficking of CTB following PIKfyve inhibition. Furthermore despite the swollen phenotype a proportion of vacuoles remained accessible to endocytosed markers suggesting direct defects in endocytosis do not underlie cell death. Many vacuoles contained amorphous intravacuolar membranes and inclusions reminiscent of autolysosomes. Consistent with this there was an increase in the level of the autophagosomal marker protein LC3-II that correlated with the onset of vacuolation and was augmented by inhibition of lysosomal proteases. However, PIKfyve inhibition in PC12s stably expressing a reporter tandem-fluorescent LC3 construct showed a selective loss of autophagosomes suggesting that de novo autophagosome formation was not upregulated. Analysis of GFP-LC3 processing in PC12 cells found an additive effect of PIKfyve inhibition and protease inhibitors on LC3-II accumulation but not GFP cleavage further suggesting that PIKfyve activity is required for the formation of fully degradative autolysosomes. Together these data indicate that PIKfyve inhibition results in dysregulated proteolysis of autophagocytosed proteins by blocking the fusion of acidified late autophagosomal compartments with the lysosome, and further suggest that alterations in the flux of material through the autophagosomal system could underlie the cell death observed upon PIKfyve inhibition in neurons.