Various sight-threatening diseases are caused by an elevated permeability of the layer of retinal microvascular endothelial cells (REC), induced by high intravitreal levels of vascular endothelial growth factor-A165 (VEGF-A165). Barrier impairment is accompanied by an altered expression of proteins restricting para- and transcellular flow, counteracted by inhibition of VEGF-A-signaling. Here we investigated whether chloroquine, an inhibitor of lysosome acidification and autophagy, can prevent VEGF-A165-induced impairment of the REC barrier. Cells were treated with 1-10 µM chloroquine ± 1.3 nM VEGF-A165 for up to four days. Barrier function was assessed by continuous cell index measurements revealing that 10 µM chloroquine partly counteracted the VEGF-A165-induced low cell index. Higher protein expression of the regulator of transcellular flow, plasmalemma vesicle-associated protein was also prevented, but not loss of tight junction protein claudin-1. Chloroquine in combination with VEGF-A165 also lowered the protein expression of differentiation markers von Willebrand factor and caveolin-1, while the abundance of a cleavage product derived from adherens junction protein vascular endothelial cadherin increased. Importantly, the prominent localization of tight junction protein claudin-5 at the plasma membrane dramatically weakened, as shown by immunofluorescence staining. Taken together, despite apparently stabilizing the barrier formed by REC, chloroquine profoundly alters the endothelial phenotype.