Abstract: Objective: In the present investigation, effect of melatonin and zafirleulast in acute lung injury model through NF-kappa beta (NF-κβ) mediated anti-inflammatory signaling pathway was studied. Materials and Methods: Acute lung injury was induced by lipopolysaccharide in mice with the animals were treated with melatonin and zafirlucast alone and combination to assess its effect on various inflammatory mediators IL-1 beta, IL-6, IL-10 and TNF-α. The effect on myeloperoxidase activity, PMN cells and the NF-κβ expressions has been evaluated in treatment groups. Results: In treatment with melatonin and zafirlucast showed amelioration of lung injury as it decreased level of proinflammatory mediators’ increase in case of lung injury. In case of combination of treatment shows potentiation of activity as level of pro-inflammatory mediators was significantly decreased. The combination of melatonin and zafirlucast significantly reduced expression of NF-κβ as compared to treatment alone in lipopolysaccharide induced acute lung injury in mice. Conclusion: In conclusion, beneficial effect of melatonin and zafirlucast in acute lung injury model whereas combination of both results in potentiation of the anti-inflammatory effect in acute lung injury.