Erythromycin (EM) has an anti-inflammatory effect that may account for its clinical benefit in the treatment of chronic inflammatory diseases such as diffuse panbronchiolitis. To investigate the mechanism of this anti-inflammatory effect, we studied the relationship between the concentration of EM and matrix metalloproteinase-9 (MMP-9) activity, which is important in cell migration. We showed that EM suppressed the gelatinolytic activity of U937 cell-derived MMP-9 by using gelatin zymography, showed that expressions of MMP-9 protein and MMP-9 mRNA were down-regulated by EM in a dose-dependent manner, and showed that U937 migration was also suppressed by EM. We also demonstrated that EM treatment suppressed the gelatinolytic activity of MMP-9 in spleen macrophages. These findings suggest that the suppressive effect of EM on MMP-9 activity is one of the anti-inflammatory mechanisms that inhibit the migration of inflammatory cells into the inflammatory site.