Acute lung injury (ALI) is a critical clinical condition with a high mortality rate. It is believed that the inflammatory storm is a critical contributor to the occurrence of ALI. Fucoxanthin is a natural extract from marine seaweed with remarkable biological properties, including antioxidant, anti-tumor, and anti-obesity. However, the anti-inflammatory activity of Fucoxanthin has not been extensively studied. The current study aimed to elucidate the effects and the molecular mechanism of Fucoxanthin on lipopolysaccharide-induced acute lung injury. In this study, Fucoxanthin efficiently reduced the mRNA expression of pro-inflammatory factors, including IL-10, IL-6, iNOS, and Cox-2, and down-regulated the NF-kappaB signaling pathway in Raw264.7 macrophages. Furthermore, based on the network pharmacological analysis, our results showed that anti-inflammation signaling pathways were screened as fundamental action mechanisms of Fucoxanthin on ALI. Fucoxanthin also significantly ameliorated the inflammatory responses in LPS-induced ALI mice. Interestingly, our results revealed that Fucoxanthin prevented the expression of TLR4/MyD88 in Raw264.7 macrophages. We further validated Fucoxanthin binds to the TLR4 pocket using molecular docking simulations. Altogether, these results suggest that Fucoxanthin suppresses the TLR4/MyD88 signaling axis by targeting TLR4, which inhibits LPS-induced ALI, and fucoxanthin inhibition may provide a novel strategy for controlling the initiation and progression of ALI.