Semaphorin3E (Sema3E) is a secreted protein that was originally implicated in the development of the nervous system. However, its role in processes other than neuronal guidance is not fully understood. Sema3E interacts with the receptor PlexinD1 with high affinity. Furthermore, differential expression of PlexinD1 with neuropilin-1 (Nrp1) and Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) determines pro-migratory or anti-migratory property of Sema3E. Recent studies demonstrated that semaphorins exhibit an inhibitory effect in most of inflammatory diseases. Among all inflammatory cells, neutrophils are an indispensable component of innate immunity and they are the foremost cells migrating to the site of inflammation. Substantial evidence indicated that the number of neutrophils is elevated in many inflammatory diseases. The aim of this study is to determine the expression pattern of Sema3E and its receptors, PlexinD1, Nrp1 and VEGFR2 in human neutrophils and to investigate the role of Sema3E on neutrophils’ migration. Here we found that isolated human neutrophils, from peripheral blood of healthy volunteers, constitutively express Sema3E and its receptors PlexinD1 and VEGFR2 at both protein and mRNA level; however, Nrp1 expression was not detected in these cells. Additionally, Sema3E display a potent ability to inhibit CXCL8/IL-8 induced neutrophils migration as determined by transwell in vitro system and microfluidic device coupled to real-time microscopy. Our data showed that Sema3E modulates the migration of neutrophils induced by the most potent chemoattractant stimuli, CXCL8/IL-8, suggesting an important regulatory role of this pathway in inflammatory diseases associated with neutrophilia.