Sepsis is an overwhelming systemic inflammatory response to microbial infections. Macrophages are the key innate immune cells that provide the first line of defence against systemic infections during sepsis. Macrophages perform multiple functions during infections such as triggering inflammation, phagocytosis of microbes, and resolution of inflammation. So far, various molecules have been shown to be involved in the regulation of macrophages in inflammatory conditions. However, recently published studies suggest that Semaphorin3E (Sema3E) plays a pivotal role in the immune function of macrophages. The exact role of Sema3E associated with macrophages function in lipopolysaccharide (LPS) induced endotoxemia is unknown. To directly address the involvement of Sema3E in macrophages, we have used Sema3e gene deletion approaches in in vivo and cell-based setups. We found that Sema3e-/- mice displayed initial transient protection from LPS-induced hypothermia. Sema3e-/- mice showed lower inducible nitric oxide synthase (iNOS) expression in peritoneal macrophages without altering the integrity of TLR-4 after LPS injection. Sema3e-/- mice exhibit a lower level of tumour necrosis factor (TNF) and interleukin-6 (IL-6) in peritoneal lavage and serum as compared to wild type (WT) littermates. Bone marrow derived macrophages (BMDMs) from Sema3e-/- mice expressed low levels of pro-inflammatory cytokines and also exhibited significantly down-regulated phosphorylation of STAT3, ERK1/2, and NF-κB, upon LPS exposure. Overall, the current study provides direct evidence that the lack of Sema3E, makes macrophages to become less responsive to LPS by disturbing LPSIII initiated signaling transduction. These findings suggest that the inhibition of Sema3E might be a novel strategy to treat conditions triggered by the excessive production of inflammatory cytokines.