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In yeast and animal cells, phosphatidylinositol‐3‐monophosphate 5‐kinases produce phosphatidylinositol (3,5)‐bisphosphate (PtdIns(3,5)P₂) and have been implicated in endomembrane trafficking and pH control in the vacuole. In plants, PtdIns(3,5)P₂ is synthesized by the Fab1 family, four orthologs of which exist in Arabidopsis: FAB1A and FAB1B, both from the PIKfyve/Fab1 family; FAB1C and FAB1D, both without a PIKfyve domain and of unclear role. Using a reverse genetics and cell biology approach, we investigated the function of the Arabidopsis genes encoding FAB1B and FAB1D, both highly expressed in pollen. Pollen viability, germination and tube morphology were not significantly affected in homozygous mutant plants. In vivo, mutant pollen fertilized ovules leading to normal seeds and siliques. The same result was obtained when mutant ovules were fertilized with wild‐type pollen. Double mutant pollen for the two genes was able to fertilize and develop plants no different from the wild‐type. At the cellular level, fab1b and fab1d pollen tubes were found to exhibit perturbations in membrane recycling, vacuolar acidification and decreased production of reactive oxygen species (ROS). Subcellular imaging of FAB1B‐GFP revealed that the protein localized to the endomembrane compartment, whereas FAB1D‐GFP localized mostly to the cytosol and sperm cells. These results were discussed considering possible complementary roles of FAB1B and FAB1D.