〔Abstract〕 Objective To investigate whether the CD151 YXXφ motif point mutant affects vascular permeability by regulating vesicle internalization and recycling. Methods CD151 knockout (KO) and wild-type (WT) mice were used. A modified Miles assay was performed to compare vascular permeability in the dorsal and postauricular skin of the two groups of mice under vascular endothelial growth factor A (VEGF-A) stimulation. The CD151 YXXφ motif point mutant YALA was constructed and transfected into human endothelial cells (EA.hy926) using an adenoviral vector. Multiple experimental groups were set up to detect the permeability of endothelial cell monolayers to FITC-dextran under baseline conditions and after VEGF-A stimulation. Western blot (WB), RT-qPCR, and immunofluorescence were used to examine the expression and distribution of VE-cadherin. The internalization rate of VE-cadherin was quantitatively compared using a cell surface biotinylation assay. Phalloidin was used to label F-actin to observe changes in cytoskeletal tension. Results The Miles assay showed that under VEGF-A stimulation, dye extravasation in the dorsal and postauricular skin of KO mice was significantly higher than that in WT mice (all P<0.05). At 60 min and 120 min after VEGF-A stimulation, the permeability of endothelial cell monolayers overexpressing the YALA mutant to FITC-dextran significantly increased (P<0.05). WB and RT-qPCR results indicated that the YALA mutant did not affect the total protein or mRNA expression levels of VE-cadherin. Immunofluorescence revealed that the YALA mutant disrupted the continuous distribution of VE-cadherin on the cell membrane and its perinuclear co-localization with CD151 under VEGF-A stimulation. The cell surface biotinylation assay confirmed that the YALA mutant significantly reduced the internalization rate of VE-cadherin (P<0.05). Cytoskeletal staining showed that the YALA mutant caused the formation of thick tension fibers in the actin cytoskeleton of endothelial cells, indicating cell contraction. Conclusion The CD151 YXXφ motif point mutation affects vesicle internalization and recycling in endothelial cells, influences the expression and internalization of VE-cadherin, and consequently affects vascular permeability.