The bioactive phenylethanoid 3,4-dihydroxyphenylethyl alcohol glycoside (DAG) is a component isolated from Sargentodoxa cuneata. The effects of DAG on acute lung injury (ALI) are largely unknown. Here, the effects of DAG on sepsis-induced ALI were investigated, and the related mechanisms were explored. Male C57BL/6 mice were used to establish a sepsis-induced ALI model. Levels of inflammatory cytokines were determined using real-time quantitative reverse transcription PCRs (qRT-PCR) and enzyme-linked immunosorbent assays (ELISAs). Pathological changes in the lung tissues were evaluated using haematoxylin and eosin (HE) staining. Mouse survival was quantified, and macrophage polarization was analyzed using flow cytometry. Our results showed that, in septic mice, pretreatment with DAG significantly improved survival, reduced histological damage in the lung, and suppressed the inflammatory response by inhibiting the activation of the NF-κB, STAT3, and p38 MAPK signaling pathways. Moreover, DAG treatment reduced the percentage of M1 macrophages in the bronchoalveolar lavage fluid (BALF) and spleen. In addition, DAG treatment decreased the production of pro-inflammatory cytokines and suppressed the activation of the NF-κB, STAT3, and p38 MAPK signaling pathways in LPS-induced MH-S cells. DAG treatment also reduced the relative abundances of M1 macrophages and M1 macrophage markers by suppressing the activation of the Notch1 signaling pathway. Thus, our results provided new insights for the development of drugs to treat ALI.