Macrolide antibiotics have immunomodulatory activities, including suppression of cytokine production, cell adhesion molecule expression, and mucin production. These immunomodulatory activities improve the symptoms of respiratory diseases associated with chronic inflammation. However, the underlying molecular mechanism(s) is not well understood yet. To address this, we prepared clarithromycin (CAM)-conjugated Sepharose and examined bound cellular proteins by proteome analysis. We identified mitochondrial proteins 4-nitrophenylphosphatase domain and non-neuronal synaptosomal associated protein 25-like protein homolog (NIP-SNAP)-1 and -2 and very long-chain acyl-CoA dehydrogenase (VLCAD) as CAM-binding proteins. Production of proinflammatory cytokines (IL-8 and IL-6) induced by lipopolysaccharides (LPSs) and Pam(3)-CSK4 in human epithelial cell lines BEAS-2B and T24 were suppressed by knockdown of NIP-SNAP -1 or -2, and partly by knockdown of VLCAD. Also, knockdown of NIP SNAP-1 or -2 in various cell lines suppressed LPS-induced expression of IL-8 and IL-6 mRNA and NF-kappa B activity. Thus, CAM suppresses NF-kappa B-mediated proinflammatory cytokine production by interacting with mitochondrial proteins, NIP-SNAP-1 and -2. (C) 2016 The Authors. Published by Elsevier Inc.