ETHNOPHARMACOLOGICAL RELEVANCE: Nauclea officinalis Pierre ex Pitard, a traditional medicinal plant cherished by the Li ethnic group, is renowned for its potent anti-allergic properties in the clinic, especially in alleviating respiratory inflammations. This plant is now extensively cultivated to harness its valuable extracts for the production of traditional cough syrup. AIM OF THE STUDY: To identify the active components responsible for its anti-allergic inflammatory properties and to elucidate the underlying mechanisms of action. METHOD: The structures of the compounds were meticulously elucidated using comprehensive spectroscopic analyses, including IR, UV, NMR, and MS, along with crystallography and ECD calculations. In vitro studies utilized IgE-induced RBL-2H3 cells to assess cell degranulation and the release of inflammatory mediators such as histamine, leukotrienes (LTs), and prostaglandins (PGs). Techniques such as the CCK-8 assay, enzyme-linked immunosorbent assay (ELISA), and western blot (WB) were employed. Additionally, molecular docking methods were used to simulate and analyze the binding interactions of small molecules with their targets. RESULT: Two novel monoterpene indole alkaloids were isolated: nauclofficine A, characterized by a unique 6/5/5/6/5 fused ring system, and nauclofficine B, which features an additional benzene-substituted dioxane moiety. Their structures were meticulously elucidated, and their biosynthetic pathways were hypothesized. Nauclofficine B exhibited significant inhibitory effects on cell degranulation and the release of inflammatory mediators such as histamine, leukotrienes (LTs), and prostaglandins (PGs) in IgE-induced RBL-2H3 cells. Further analysis using western blot revealed that it significantly modulated the expression of key proteins in the IgE-mediated inflammatory signaling pathway. Molecular docking experiments suggested that its potential multi-target proteins include Lyn, FcεR, Syk, and p-Gab2, which were subsequently verified. CONCLUSIONS: These results suggested that the wood of N. officinalis contains structurally novel alkaloids with anti-allergic inflammatory activity. The absolute configuration of the compound was accurately and reliably deduced, and the biosynthetic pathway was reasonably inferred. Their primary mechanism of action involves targeting multiple key proteins in the IgE-mediated inflammatory signaling pathway, including Lyn, FcεR, Syk, and p-Gab2.