The aim of this study was to elucidate whether the membrane nanodomain protein Flot1 is involved in vesicular transport pathways and regulation of the P-type H-ATPase content in plasma membrane of under salt stress. Transmission electron microscopy revealed changes in the endosomal system of root cells due to knockout mutation SALK_205125C (). Immunoblotting of the plasma membrane-enriched fractions isolated from plant organs with an antibody to the H-ATPase demonstrated changes in the H-ATPase content in plasma membrane in response to the mutation and salt shock. Expression levels of the main H-ATPase isoforms, and , as well as endocytosis activity of root cells determined by endocytic probe FM4-64 uptake assay, were unchanged in the mutant. We have shown that Flot1 participates in regulation of the H-ATPase content in the plasma membrane. We hypothesized that Flot1 is involved in both exocytosis and endocytosis, and, thus, contributes to the maintenance of cell ion homeostasis under salt stress. The lack of a pronounced phenotype under salt stress conditions may be due to the assumed ability of to switch vesicular transport to alternative pathways. Functional redundancy of Flot proteins may play a role in the functioning of these alternative pathways.