The objectives of the present study were to determine if IQGAP1 associates with components of the endothelial adherens junction and if it affects endothelial barrier function. In human umbilical vein endothelial cells (HUVECs), soluble IQGAP1 associated with VE-cadherin and the catenins, b, y, and a, but not N-cadherin. Gene silencing of IQGAP1 by transfection of small interfering RNA (siRNA) with oligofectamine maintained a higher endothelial electrical resistance in HUVECs as compared to transfection of a scrambled siRNA. Reduction of IQGAP1 induced an increase and a decrease, respectively, in the protein levels of VE-cadherin and N-cadherin. More VE-cadherin and less N-cadherin were associated with p120- and B -catenins in IQGAP1 knockdown cells. Furthermore, more insoluble (actin-associated) VE-cadherin was localized at intercellular junctions and less insoluble N-cadherin was present in the cell. These findings suggest that a reduction of IQGAP1 positively influences the endothelial barrier by increasing the protein level of VE-cadherin and the interaction of VE-cadherin with the actin cytoskeleton. The increased interaction with the catenins, B and p120, possibly resulting from the decrease in N-cadherin, may explain the increase in VE-cadherin.