Phosphatidylinositol (PI) is a constituent of biomembranes and a precursor of all phosphoinositides (PIPs). A prominent characteristic of PI is that its sn‐2 position is highly enriched in polyunsaturated fatty acids (PUFAs), such as arachidonic acid or eicosapentaenoic acid. However, the biological significance of PUFA‐containing PI remains unknown. We previously identified Caenorhabditis elegans (C. elegans) mboa‐7 as an acyltransferase that incorporates PUFAs into the sn‐2 position of PI. In this study, we performed an RNAi enhancer screen against PI kinases and phosphatases using mboa‐7 mutants that have a reduced PUFA content in PI. Among the genes tested, knockdown of vps‐34, a catalytic subunit of class III PI 3‐kinase that produces PI 3‐phosphate (PI3P) from PI, caused severe growth defects in mboa‐7 mutants. In both vps‐34 RNAi‐treated wild‐type worms and mboa‐7 mutants, the size of PI3P‐positive early endosomes was significantly decreased. We also performed an RNAi enhancer screen against PI3P‐related genes and found that, like knockdown of vps‐34, knockdown of autophagy‐related genes caused severe growth defects in mboa‐7 mutants. Finally, we showed that autophagic clearance of protein aggregates is impaired in mboa‐7 mutants. Taken together, these results suggest that the PUFA chain in PI has a role in some PI3P signaling.