Background: Bicyclomycin (BCM) is a unique antibiotic that selectively inhibits the bacterial transcription termination factor, Rho. Recent genomic analyses have revealed widespread distribution of BCM biosynthetic gene clusters across diverse bacterial species, suggesting additional biological functions beyond antimicrobial activity. We hypothesized that BCM may possess anti-inflammatory properties that contribute to its evolutionary conservation. Methods: We investigated the anti-inflammatory effects of BCM using LPS-stimulated RAW 264.7 macrophages. Cell viability was assessed using MTT assays, and inflammatory mediator production (nitric oxide, TNF-α, IL-6, and IL-1β) was measured using colorimetric and ELISA-based assays. Mechanistic insights were explored through pathway analysis. Results: BCM demonstrated dose-dependent anti-inflammatory activity in LPS-stimulated macrophages. Treatment with BCM (25-100 μM) significantly reduced production of nitric oxide, TNF-α, IL-6, and IL-1β compared to LPS-only controls. These effects occurred at concentrations that maintained cell viability above 100 %. Pathway analysis suggested involvement of NF-kB-mediated signaling cascades. Conclusions: This study provides the first evidence for anti-inflammatory properties of bicyclomycin, revealing a previously unrecognized therapeutic potential. The widespread distribution of BCM biosynthetic gene clusters may reflect evolutionary pressure for dual antimicrobial and anti-inflammatory functions. These findings suggest new therapeutic applications for BCM and highlight the importance of investigating pleiotropic effects of antibiotics with unique mechanisms of action.