Author summary IAV is one of the most common respiratory pathogens, leading to acute lung injury and a high morbidity rate. Melatonin has important regulatory roles in modulating immune responses during acute inflammatory conditions. It is of great significance to explore the protection effect of melatonin against the H1N1-induced pulmonary injury. Here, we found that melatonin had essential impacts on protection against IAV infection in mice. For the first time, we proved that melatonin had the capacity to suppress the activation of mast cells and the associated inflammatory response during H1N1 infection. The molecular mechanisms involved melatonin down-regulation of gene expression for the HIF-1 pathway mediated by MT2 and inhibition of proinflammatory cytokine release from mast cells, ultimately, leading to a reduction in the migration and activation of the macrophages and neutrophils in the lung tissue. Consequently, melatonin suppressed apoptosis of alveolar epithelial cells and the lung injury caused by H1N1 infection. Our findings highlight a novel mechanism to protect against the H1N1-induced pulmonary injury, and also provide insights into the new strategies to fight IAV infections.