Objective: To investigate the effects of the murine inhibitory vascular endothelial growth factor (VEGF, rVEGF164b), we generated an adenoviral vector encoding rVEGF164b, and examined its effects on endothelial barrier, growth, and structure. Method: Mouse vascular endothelial cells (MVEC) proliferation was determined by an MTT assay. Barrier of MVEC monolayers was measured by trans-endothelial electrical resistance (TEER). Reorganization of actin and zonula occludens-1 (ZO-1) were determined by fluorescent microscopy. Results: Mouse venous endothelial cells treated with murine VEGF-A (VEGF-A) (50 ng/mL) increased proliferation (60.7 ± 0.1%) within 24 hours (p < 0.05) and rVEGF164b inhibited VEGF-A-induced proliferation. TEER was significantly decreased by VEGF-A (81.7 ± 6.2% of control). Treatment with rVEGF164b at 50 ng/mL transiently reduced MVEC barrier (p < 0.05) at 30 minutes post-treatment (87.9 ± 1.7% of control TEER), and returned to control levels by 40 minutes post-treatment. Treatment with rVEGF164b prevented barrier changes by subsequent exposure to VEGF-A. Treatment of MVECS with VEGF-A reorganized F-actin and ZO-1, which was attenuated by rVEGF164b. Conclusions: VEGF-A may dysregulate endothelial barrier through junctional cytoskeleton processes, which can be attenuated by rVEGF164b. The VEGF-A stimulated MVEC proliferation, barrier dysregulation, and cytoskeletal rearrangement. However, rVEGF164b blocks these effects, therefore it may be useful for regulation studies of VEGFA/VEGF-R signaling in many different models.