Monoammonim glycyrrhizate (MAG) derived from licorice has been shown to have anti-inflammatory properties. Chemokines are vital inflammatory mediators that are involved with endothelial damage from leukocyte infiltrates in various inflammatory skin diseases. In this study, we investigated the anti-inflammatory effects and mechanisms of MAG on tumor necrosis factor-α (TNF-α) induced chemokine production in a human dermal microvascular endothelial cell line (HMEC-1). HMEC-1 cells were treated with TNF-α, with or without MAG. The results showed that MAG suppressed TNF-α-induced chemokine (including CXCL8, CX3CL1, and CXCL16) mRNA expression in HMEC-1 cells, in a dose-dependent manner, and reduced the secretion of these chemokines in culture supernatant. Moreover, endothelial activation in the presence of MAG blocked the chemotactic activities of TNF-α-stimulated HMEC-1 cell supernatant on the migration of primary neutrophils and primary monocytes. In addition, Western blot and immunofluorescence data revealed that MAG inhibited nuclear translocation of nuclear factor-κB p65 (NF-κB p65). It is the first report to demonstrate that MAG suppresses TNF-α-induced chemokine production in HMEC-1 cells, and that the mechanism may be inhibiting the translocation of NF-κB p65 into the nucleus to prevent the starting of inflammatory signaling pathway. Our results revealed that MAG is a potential anti-inflammatory agent capable of improving inflammatory skin diseases.