Acute lung inflammation (ALI) poses a significant global health challenge. Conventional synthetic anti-inflammatory drugs often show limited efficacy against ALI and may cause side effects. Natural fucoidans exhibit promising anti-inflammatory properties, but their specific effects and mechanisms in ALI remain unclear. In this study, we characterized the structural features of Apostichopus japonicus-derived fucoidan AJ2-I using methylation analysis, 1D/2D nuclear magnetic resonance, and chemical assays. We then evaluated its anti-ALI effects in lipopolysaccharide-challenged C57BL/6J mice and human A549 cells. Structural analysis revealed that AJ2-I primarily consists of [→3)-Fucp-(1→] residues with C4 branching, while sulfate groups were predominantly substituted at C2 and C4 positions. Notably, this fucoidan contains unique glycosyls—→4)-β-D-GalpA-6-O-Et-(1→ and →4)-β-D-GalpA-6-OMe-(1→—distinguishing it from previously reported A. japonicus polysaccharides. Functionally, AJ2-I significantly suppressed pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) in both in vivo and in vitro models. Mechanistically, it inhibited Toll-like receptor 2/4 to suppress mitogen-activated protein kinase and phosphoinositide-3 kinase/protein kinase B signaling pathways, thereby blocking the activation of key transcription factors, including nuclear factor kappa-B and c-Jun/activator protein-1. These findings elucidate the protective role of A. japonicus-derived fucoidan AJ2-I in ALI and uncover its underlying molecular mechanisms, supporting the potential use of fucoidans in treating lung inflammation.