Orange-spotted grouper (Epinephelus coioides) is among the most economically important of all fish species farmed in Asia. This species expresses an antimicrobial peptide called epinecidin-1 (EPI), which is considered to be a host defense factor due to its strong bacterial killing activity. Antimicrobial peptides usually possess both bacterial killing and immunomodulatory activity, however, the modulatory activity of EPI on Gram-positive bacterial lipoteichoic acids (LTA)-induced inflammation has not been previously reported. In this study, we found that EPI effectively suppressed LTA-induced production of proinflammatory factors in macrophages. Mechanistically, EPI attenuated LTA-induced inflammation by inhibiting Toll-like receptor (TLR) 2 internalization and subsequent downstream signaling (reactive oxygen species, Akt, p38 and Nuclear factor κB). However, protein abundance of TLR2 was not altered by EPI or LTA. Taken together, our findings reveal for the first time that EPI possesses inhibitory activity toward LTA-induced inflammation in macrophages. •Orange-spotted grouper antimicrobial peptide epinecidin-1 inhibits lipoteichoic acid-induced inflammation in macrophages.•Epinecidin-1 inhibits Toll-like receptor 2 internalization and subsequent signaling.•Epinecidin-1 has pharmacological potential as an anti-infective therapy.