Neutrophil extracellular traps (NETs) play an important role in the formation of immunothrombosis. However, how vascular endothelial cells mediate the formation of NETs has not been fully understood. We stimulated neutrophils firmly attached on the endothelial cell surface intercellular adhesion molecule-1 (ICAM-1) with lipopolysaccharide (LPS) or phorbol-12-myristate-13-acetate (PMA) for 4 h, then labeled NETs-DNA with Sytox green dye and the formation of NETs was observed by fluorescent microscopy. The area and fluorescence intensity of NETs-DNA were analyzed to quantify the formation of NETs. The results showed that both PMA and LPS were able to induce firmly adhered neutrophils on ICAM-1 to produce NETs. NETs induced by PMA were independent of neither β2 integrin lymphocyte function-associated antigen-1 (LFA-1) nor macrophage antigen complex-1 (Mac-1). In contrast, LPS-stimulated NETs were mediated by Mac-1 integrin, but not by LFA-1. After inhibition of actin filaments or Talin-1, the formation of NETs irrespective of the stimulus was significantly reduced. This study reveals the mechanism of the direct interaction between neutrophils and endothelial cells to produce NETs under inflammatory conditions, providing a new theoretical basis for the treatment of related diseases and the development of new drugs.