With the rapidly expanding use of nanoparticles in research and industry, the need to understand their potential for risk to population health is imperative. Current information on the effects of nanoparticles on living systems is limited, and this lack of knowledge has led to a global effort to define risk and improve the safety of nanoparticles. In this work, we elucidated the impact of gold nanoparticles (GNPs) on cell mechanical behavior, barrier function, and cell-cell junctions as a critical step towards closing the present knowledge gap. We found tested nanoparticles affected the homeostasis of endothelial cells by disturbing the integrity of cell-cell junctions and cells mechanical properties. In addition, dark field microscopy showed that GNPs can adhere to endothelial cells or be taken up by endothelial cells easily; colorimetric assay for cell viability and reactive oxygen species assay showed the toxicity of GNPs is size dependent and surface modification dependent. These findings may have important implications regarding the safety issue as nanoparticles are widely used in biomedical applications and our daily life.