Luteolin is able to inhibit the adhesion of neutrophils to microvascular endothelial cells (MEC) and plays an anti-inflammatory role through regulating vascularScellSadhesion molecule (VCAM)-1 in MEC. The inhibitory mechanism is related to three signaling pathways: mitogen activated protein kinase (MAPK), nuclear factor-kappa B (NF-κB)/IκB, and phosphatidylinositol-3-kinase (PI3K) /Akt pathways. The pathways (MAPK and NF-κB/IκB) are involved in the positive regulation of VCAM-1, and the PI3K/Akt pathway participates in the negative regulation. We determined the effects of luteolin on phosphorylation of p38 MAPK, p65 NF-κB, and p85 PI3K, which are key proteins in the mentioned pathways, respectively. Luteolin promotes the phosphorylation of p38 MAPK at different time intervals (30 s, and 1 min) and p85 PI3K (30 s, 1 min, and 5 min); but significantly inhibits the phosphorylation of p65 NF-κB (30 s, 1 min, 5 min, and 30 min) in MEC. As our results show, inhibition of p38 MAPK pathway induces downregulation of VCAM-1. Furthermore, inhibition of the phosphorylation of p38 MAPK via SB203580, a p38 MAPK inhibitor, also downregulates VCAM-1, suggesting that the regulation of VCAM-1 in the presence of luteolin in MEC is independent upon the p38 MAPK phosphorylation. This is to say luteolin regulates VCAM-1 expression in MEC through either inhibiting p65 NF-κB phosphorylation or promoting p85 PI3K phosphorylation. This work may provide an insight for the molecular mechanism in the anti-inflammatory effect of luteolin.