This paper explored the underlying molecular mechanisms by which hydroxytyrosol exerts its anti-inflammatory effects in a murine model of acute lung injury (ALI) by up-regulating autophagy. LPS-induced cytokine activity, inflammatory factor levels, sirtuin (SIRT1/3/6) expression, mitogen-activated protein kinase (MAPK) activation, and autophagy marker expression in ALI-mice were examined by western blotting and staining methods. Molecular docking between HT and SIRT and MAPK was studied with a Sybyl/Surflex module. The results showed that LPS-stimulated SIRT inhibition, MAPK phosphorylation, and autophagy suppression were all notably abolished by HT administration. HT treatment significantly attenuated pulmonary edema and inflammatory cell infiltration into lung tissues, accompanied by decreased lung W/D ratios, protein concentrations, and inflammatory cell levels in BAL fluid. LPS driven release of inflammatory mediators, including TNF-α, IL-1β, IL-6, IL-10, and MCP-1, was strongly regulated by HT.