Interleukin-17A (IL-17A) is an important pro-inflammatory cytokine that regulates leukocyte mobilization and recruitment. To better understand how IL-17A controls leukocyte trafficking across capillaries in the peripheral blood circulation, we used primary human dermal microvascular endothelial cells (HDMEC) to investigate their secretory potential and barrier function when activated with IL-17A and TNFα. Activation by TNFα and IL-17A causes phosphorylation of p38 as well as IκBα whereby NFκB subsequently becomes phosphorylated, a mechanism that initiates transcription of adhesion molecules such as E-selectin. Members of the neutrophil-specific GRO-family chemokines were significantly up-regulated upon IL-17A stimulation on the mRNA and protein level, whereas all tested non-neutrophil-specific chemokines remained unchanged in comparison. Moreover, a striking synergistic effect in the induction of granulocyte colony-stimulating factors (G-CSF) was elicited when IL-17A was used in combination with TNFα, and IL-17A was able to significantly augment the levels of TNFα-induced E-selectin and ICAM-1. In accordance with this observation, IL-17A was able to markedly increase TNFα-induced neutrophil adherence to HDMEC monolayers in an in vitro adhesion assay. Using a trans-well migration assay with an HDMEC monolayer as a barrier, we here show that pre-stimulating the endothelial cells with TNFα and IL-17A together enhances the rate of neutrophil transmigration compared to TNFα or IL-17A alone. These results show that IL-17A and TNFα act in cooperation to facilitate neutrophil migration across the endothelial cell barrier. In addition, the synergistic actions of IL-17A with TNFα to secrete G-CSF appear to be important for mobilizing neutrophils from the bone marrow to the blood stream.