Lipid kinase FORMATION of APLOID and BINUCLEATE CELLS 1 (FAB1/PIKfyve) is a key enzyme that generates PI(3,5)P2. PI(3,5)P2 plays an important role in regulating membrane trafficking in yeast and animal cells, but its function in plants remains poorly understood. Here we study the functional role of PI(3,5)P2 via analysis of phenotypes of Arabidopsis FAB1 T-DNA knockout lines. The Arabidopsis FAB1 family contains four orthologous genes: FAB1A, FAB1B, FAB1C, and FAB1D. The fab1a/b double mutant showed a complete male gametophyte lethal phenotype. fab1b/c/d mutant was successfully isolated. Genetic analyses showed that the loss of FAB1B, FAB1C and FAB1D function disrupts root hairs elongation. Further pharmacological analysis showed that the FAB1-specific inhibitor YM201636 inhibited the root hairs growth. In addition, the transcription level of DR5-GFP, an indicator of auxin expression and distribution, was downregulated in single mutation of FAB1B, FAB1C and FAB1D. Moreover, triple mutant phenotypes (short root hairs) were partially rescued by exogenous application of auxin analog 2,4-D and NAA. However, the mutant’s sensitivity to 1-NOA (auxin influx inhibitor) and TIBA (auxin efflux inhibitor) in root hair elongation assay is identical to that of the wild-type. Furthermore, loss of FAB1B, FAB1C and FAB1D function reduces the level of reactive oxygen species (ROS) and affects actin expression in root hairs. Taken together, the FAB1B/C/D affects Arabidopsis root hair elongation by regulating auxin distribution, levels of ROS, and actin expression.