Phosphoinositides and their metabolizing enzymes are involved in Aβ42 metabolism and Alzheimer's disease pathogenesis. In yeast and mammals, Eighty-five requiring 3 (EFR3), whose Drosophila homolog is Rolling Blackout (RBO), forms a plasma membrane-localized protein complex with phosphatidylinositol-4-kinase Type IIIα (PI4KIIIα) and a scaffold protein to tightly control the level of plasmalemmal phosphatidylinositol-4-phosphate (PI4P). Here, we report that RBO binds to Drosophila PI4KIIIα, and that in an Aβ42-expressing Drosophila model, separate genetic reduction of PI4KIIIα and RBO, or pharmacological inhibition of PI4KIIIα ameliorated synaptic transmission deficit, climbing ability decline, premature death, and reduced neuronal accumulation of Aβ42 Moreover, we found that RBO-PI4KIIIa downregulation increased neuronal Aβ42 release and that PI4P facilitated the assembly or oligomerization of Aβ42 in/on liposomes. These results indicate that RBO-PI4KIIIa downregulation facilitates neuronal Aβ42 release and consequently reduces neuronal Aβ42 accumulation likely via decreasing Aβ42 assembly in/on plasma membrane. This study suggests the RBO-PI4KIIIα complex as a potential therapeutic target and PI4KIIIα inhibitors as drug candidates for Alzheimer's disease treatment.SIGNIFICANCE STATEMENT Phosphoinositides and their metabolizing enzymes are involved in Aβ42 metabolism and Alzheimer's disease pathogenesis. Here, in an Aβ42-expressing Drosophila model, we discovered and studied the beneficial role of downregulating RBO or its interacting protein PI4KIIIα-a protein that tightly controls the plasmalemmal level of PI4P-against the defects caused by Aβ42 expression. Mechanistically, RBO-PI4KIIIα downregulation reduced neuronal Aβ42 accumulation, and interestingly increased neuronal Aβ42 release. This study suggests the RBO-PI4KIIIα complex as a novel therapeutic target, and PI4KIIIα inhibitors as new drug candidates.