Despite extensive studies on cellular responses to activation of Toll-like receptor-4 (TLR-4), it is not evident weather its activation affects gene expression of interleukin-8 (IL-8) in vascular smooth muscle cells (VSMCs). Therefore, this study has investigated whether and how TLR-4 influences IL-8 expression in VSMCs. Exposure of aortic smooth muscle cells to TLR-4 agonistic lipopolysaccharide (LPS) not only enhanced release of IL-8 protein but also induced IL-8 gene transcript via promoter activation. The LPS-induced activation of IL-8 promoter was attenuated by dominant-negative MKK1, but not by dominant-negative MKK3. The promoter activation was also impaired by dominant negative CCAAT/enhancer binding protein (C/EBP), IkappaB, and dominant negative c-Jun. In comparison with the mutation of the AP-1 binding site, the mutation of NF-kappaB site and C/EBP binding site in the IL-8 promoter region more significantly impaired the promoter activation. Moreover, both promoter activity and release of IL-8 were inhibited by U0126 and curcumin, but not by SB202190, epigallocatechin 3-gallate and resveratrol. The present study reports that TLR-4-agonistic LPS upregulates IL-8 at the transcriptional and post-translational level in VSMCs, and that ERK1/2, NF-kappaB, and C/EBP play major roles in the upregulation of IL-8.