Vascular inflammatory injury leads to vascular endothelial dysfunction, thereby resulting in a variety of cardiovascular diseases (CVDs). Thus, attenuating vascular inflammatory injury has great significance for the prevention and treatment of CVDs. In China, Shenmai formula, a well-known ancient Chinese prescription, has been widely used to treat CVDs, such as coronary atherosclerosis and viral myocarditis. In vivo study has demonstrated that the optimal combination of 3 major active components from Shenmai formula, panaxadiol and panaxatriol type saponins and ophioponins, in a ratio of 1:2:2 might exert significant cardioprotective effects and anti-inflammatory activities. The aim of this study was to investigate whether the combination may exert anti-inflammatory effects on lipopolysaccharide-induced inflammatory injury in cardiac microvascular endothelial cells by blocking nuclear factor-kappa B (NF-κB) pathway. We found that the combination could exert anti-inflammatory effects by inhibiting the mRNA and protein expression of interleukin-1, interleukin-6, tumor necrosis factor alpha, and intercellular adhesion molecule, as well as reducing the lactate dehydrogenase content in lipopolysaccharide-injured cardiac microvascular endothelial cells supernatant. Further experiments showed that the combination could suppress the NF-κB p65 expression and IκBα phosphorylation in these cells. These findings suggested that the combination inhibits vascular inflammatory injury by blocking NF-κB pathway, which proves a new molecular mechanism of the Shenmai formula to treat CVDs.