Ethnopharmacological relevanceThe fruits of Ligustrum lucidum (FLL) W.T. Aiton (Oleaceae) is included in the 2020 “Chinese Pharmacopoeia” and is widely used in traditional Chinese medicine as a tonic. In recent years, FLL has been reported to improve immune function, but the bioactive compounds and mechanisms of FLL remain poorly characterized. Aim of the studyTo identify FFL compounds with strong immune activity and explore their molecular mechanisms. Materials and methodsThe phagocytic activity of RAW264.7 macrophages and proliferation activity of spleen lymphocytes were used to guide the isolation of bioactive compounds from FLL extracts. Lymphocyte subpopulations, Ca2+ concentrations, and surface molecule expression were analyzed using flow cytometry. Cytokine secretion was examined using ELISA. FITC-OVA uptake was observed using fluorescence microscopy. NF-κB activation was analyzed using western blotting. ResultsThe extraction and isolation produced ten compounds, namely oleuropeinic acid, nuezhenide, isonuezhenide, salidroside, isoligustrosidic acid, ligulucidumosides A, 8(E)-nuezhenide, hydroxytyrosol, oleuropein, and p-hydroxyphenethyl 7-β-D-glucosideelenolic acid ester were isolated and identified from FLL-Bu-30%. Immunoactivity experiments showed that hydroxytyrosol had the strongest macrophage phagocytotic and lymphocyte proliferation-promoting activities. Further studies showed that hydroxytyrosol could significantly enhance lymphocyte subsets CD3+, CD4+/CD8+, and CD3+CD4−CD8−, promote IL-4, IFN-γ, and TNF-α secretion, and increase intracellular Ca2+ concentrations. In addition, the results from RAW264.7 macrophages showed that hydroxytyrosol increased FITC-OVA uptake, induced TNF-α and IL-1β production, upregulated MHC-II, CD80, and CD86 expression, promoted cytoplasmic IκB-α degradation, and increased nuclear NF-κB p65 levels. ConclusionOur study provides substantial evidence regarding the mechanism of the immunomodulatory effects of compounds from FLL.