Antimicrobial peptides (AMPs) are attractive alternatives to antibiotics. Due to their immune modulatory properties, AMPs are at present emerging as promising agents for controlling inflammatory-mediated diseases. In this study, anti-inflammatory potential of an antimicrobial peptide, KLK (KLKLLLLLKLK) and its analogs was evaluated in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. The results herein demonstrated that KLK peptide as well as its analogs significantly inhibited the pro-inflammatory mediator nitric oxide (NO), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) production in LPS-stimulated RAW 264.7 macrophages in dose-dependent manners, and such inhibitory effects were not due to direct cytotoxicity. When considering inhibition potency, KLK among the test peptides exhibited the most effective activity. The inhibitory activity of KLK peptide also extended to include suppression of LPS-induced production of prostaglandin E2 (PGE2). KLK significantly decreased mRNA and protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) as well as mRNA expression of IL-1β and TNF-α. Moreover, KLK inhibited nuclear translocation of nuclear factor-κB (NF-κB) p65 and blocked degradation and phosphorylation of inhibitor of κB (IκB). Taken together, these results suggested that the KLK peptide inhibited inflammatory response through the down-regulation of NF-κB mediated activation in macrophages. Since peptide analogs with different amino acid sequences and arrangement were investigated for their anti-inflammatory activities, the residues/structures required for activity were also discussed. Our findings therefore proved anti-inflammatory potential of the KLK peptide and provide direct evidence for therapeutic application of KLK as a novel anti-inflammatory agent.