Temporin-1CEa, which is isolated from the skin secretions of the Chinese brown frog Rana chensinensis, exhibits broad-spectrum antimicrobial activity against gram-positive and gram-negative bacteria and antitumor activity. LK2(6) and LK2(6)A(L) are the analogs of temporin-1CEa obtained by replacing amino acids and displayed an improved anticancer activity. In the present study, the anti-inflammatory activity and mechanism of action of temporin-1CEa and its analogs LK2(6) and LK2(6)A(L) in lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophages were investigated. The results showed that temporin-1CEa and its analogs decreased the production of the cytokines tumor necrosis factor-α and interleukin-6 by inhibiting the protein expression of nuclear factor-κB and mitogen-activated protein kinase and the MyD88-dependent signaling pathway. Isothermal titration calorimetry studies revealed that temporin-1CEa, LK2(6) and LK2(6)A(L) exhibited binding affinities to LPS, an important inflammatory inducer, with Kd values of 0.1, 0.03 and 0.06 μM, respectively. Circular dichroism and zeta potential experiments showed that temporin-1CEa and its analogs interacted with LPS by electrostatic binding between the positively charged peptides and negatively charged LPS, resulting in the neutralization of LPS toxicity.