Stylissatin A (SA) is a cyclic heptapeptide isolated from the marine sponge Stylissa massa. SA shows anti-inflammatory activity against lipopolysaccharide (LPS)-stimulated murine RAW264.7 macrophage cells, but the detailed mechanism of action remains unclear. Here we report that D-Tyr1-tBuSA, a more potent SA derivative, inhibited production of the proinflammatory cytokines Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in LPS-stimulated RAW264.7 cells (EC50 = 1.4 and 5.9 μM, respectively). This compound also inhibited the LPS-stimulated expression of inducible nitric oxide synthase (iNOS) at 20 μM. Using a biotin derivative of SA, acyl-CoA dehydrogenase long chain (ACADL) was identified as a target protein of SA and its derivatives. It is proposed that SA and its derivatives might suppress the β-oxidation of fatty acids by ACADL, and the accumulation of fatty acids on macrophages would inhibit the nuclear factor-kappa B (NF-κB) signaling pathway and iNOS expression to show anti-inflammatory activity. Our research might provide a new mechanism of inflammation in macrophages, and contribute to the development of treatments for inflammatory diseases.