Tumor necrosis factor alpha (TNF-α) is a critical cytokine that is involved in systemic inflammatory response and contributes to the activation of the pro-inflammatory phenotype of the endothelium. In the present study, effects of TNF-α on morphology and elasticity of endothelium in relation to the production of NO and actin fiber reorganization were analyzed in human dermal microvascular endothelial cells. The cells were incubated in MCDB medium solution and stimulated with [Formula: see text] of TNF-α. Atomic force microscopy measurements have enabled characterization of cell morphology and elastic properties in physiological conditions. The spectrophotometric Griess method was applied to estimate nitric oxide (NO) production of the cells. We demonstrated that TNF-α-induced changes in elasticity of endothelium anti-correlate with NO production and are associated with the reorganization of actin cytoskeleton.