Apart from direct bacterial killing, antimicrobial host defence peptides (HDPs) exert various other biological activities that also include modulation of immune responses to infection. The bovine cathelicidin BMAP-28 has been extensively studied with regard to its direct antibacterial activity while little is known about its effects on immune cell function. We have investigated its ability to affect inflammatory pathways and to influence the proinflammatory response induced by LPS in RAW 264.7 macrophages, in terms of modulation of TLR4 activation and cytokine gene induction. BMAP-28 on its own elicited ERK1/2, p38 and NF-κB activation leading to upregulation of IL-1β gene expression in these cells, suggesting it has the capacity to activate selected cellular pathways through direct effects on macrophages. As expected based on its in vitro LPS-binding properties, BMAP-28 blocked LPS-induced cytokine gene expression when added to the cell culture in combination with LPS. However it enhanced the induction of IL-1β and IL-6 genes and suppressed that of IFN-β when added prior to or following LPS stimulation over a 30-60 min time interval, or when co-administered with taxol as another TLR4 stimulant. It did not inhibit the expression of IFN-β induced by the TLR3 ligand poly(I:C). Overall these results, and the fact that BMAP-28 increased the LPS-stimulated activation of NF-κB while diminishing that of IRF-3, suggest that the peptide potentiates the early TLR4-mediated proinflammatory cytokine response while inhibiting the TLR4/TRAM/TRIF signaling pathway leading to IRF-3 activation and IFN-β gene expression. Using a TLR4-specific antibody we also found that BMAP-28 decreased the LPS-induced internalization of surface TLR4 required for initiating the TRAM/TRIF signaling pathway, which provides a mechanism for the inhibitory effect of the peptide on the TLR4/TRAM/TRIF pathway.