Background and Purpose Airway inflammation in cystic fibrosis (CF) patients is characterized by accumulations of neutrophils in the airway and T cells in bronchial tissue, with activation of platelets in the circulation. CF patients are routinely treated with systemic or inhaled tobramycin for airway infection with Pseudomonas aeruginosa. Clinical trials have indicated an anti-inflammatory effect of tobramycin beyond its bactericidal activity. Here, we investigate the anti-inflammatory properties of tobramycin in vitro and consider if these relate to the ability of tobramycin to bind copper, which is elevated in blood and sputum in CF. Experimental Approach A copper–tobramycin complex was synthesized. The effect of tobramycin and copper–tobramycin on neutrophil activation and migration of T cells and neutrophils across human lung microvascular endothelial cells in response to thrombin-activated platelets were investigated in vitro. Tobramycin uptake was detected by immunocytochemistry. Intracellular reactive oxygen species were detected using the fluorescent indicator, 2′,7′-dichlorofluorescein diacetate (DCFDA). Neutrophil superoxide, hydrogen peroxide and neutrophil elastase activity were measured using specific substrates. Copper was measured using atomic absorption spectroscopy. Key Results Tobramycin and copper–tobramycin were taken up by endothelial cells via a heparan sulphate-dependent mechanism and significantly inhibited T-cell and neutrophil transendothelial migration respectively. Copper–tobramycin has intracellular and extracellular superoxide dismutase-like activity. Neutrophil elastase inhibition by α1-antitrypsin is enhanced in the presence of copper–tobramycin. Tobramycin and copper–tobramycin are equally effective anti-pseudomonal antibiotics. Conclusions and Implications Anti-inflammatory effects of tobramycin in vivo may relate to the spontaneous formation of a copper–tobramycin complex, implying that copper–tobramycin may be more effective therapy.