Objective: Brain microvascular endothelial cells are integral components of the blood-brain barrier and play a role in protecting the brain from invading microbes. CXC motif chemokine ligand 1 (CXCL1) induces the chemotaxis of neutrophils, and neutrophils are important in host defense in the brain. However, dysregulated neutrophil infiltration leads to brain diseases. Toll-like receptor 3 (TLR3) is a pattern recognition receptor that recognizes viral double-stranded RNA (dsRNA). The aim of this study was to investigate the effect of an TLR3 agonist on the expression of CXCL1 in brain vascular endothelial cells. Methods: hCMEC/D3 human cerebral microvascular endothelial cells were cultured and treated with polyinosinic-polycytidylic acid (poly IC), a potent synthetic dsRNA agonist for TLR3. The production of CXCL1 mRNA and protein was assessed by real-time RT-PCR and ELISA. The expression of CXCL1 was compared with that of CXCL8. The effect of pretreatment of cells with a NF-κB inhibitor (SN50), a p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125), an interferon (IFN) regulatory factor 3 inhibitor (MRT67307), and an anti-type I IFN-neutralizing antibody mixture was examined. Phosphorylation of p38 was examined using Western blotting. Results: Treating cultured hCMEC/D3 human cells with poly IC induced the expression of CXCL1 as well as another chemokine CXCL8. Pretreatment of cells with SN50, SB203580, and SP600125 decreased the induction of CXCL1 by poly IC. However, it was not affected by MRT67307 or by an anti-type I IFN-neutralizing antibody mixture. Pretreatment of cells with SN50 decreased the poly IC-induced phosphorylation of p38. Conclusions: Poly IC induces the expression of CXCL1 in hCMEC/D3 cells. NF-κB, p38 MAPK, and JNK are involved in this reaction. There is a cross-talk between NF-κB and p38, and NF-κB partially regulates phosphorylation of p38. CXCL1 produced by brain microvascular endothelial cells may contribute to the brain’s defense against viral infection and various neurological diseases associated with neutrophil accumulation.