〔Abstract〕 Objective To investigate the mechanism by which icariin (ICA) inhibits the invasion and metastasis of human triple-negative breast cancer (TNBC) cells via downregulation of the transforming growth factor-β/Smad (TGF-β/Smad) signaling pathway. M ethodsTNBC cells (MDA-MB-231 and MDA-MB-468) were cultured in vitro and divided into four groups: an experimental group treated with 15 μmol/L ICA; a model group treated with 10 μmol/L TGF-β receptor inhibitor LY2109761; a combination group (LY2109761+ICA) treated with both 15 μmol/L ICA and 10 μmol/L LY2109761; and a control group. Cell proliferation, migration, and invasion were assessed using CCK-8, colony formation, 5-ethynyl-2′-deoxyuridine (EdU), wound healing, and Transwell assays. The expression levels of epithelial-mesenchymal transition (EMT)-related proteins, as well as TGF-β1, Smad2, and phosphorylated Smad2 (P-Smad2) were detected by immunofluorescence and Western blot. ResultsCCK-8 results showed that cell proliferation decreased gradually with increasing concentrations of ICA ( P<0.05). Colony formation and EdU assays indicated significantly inhibited proliferation in the ICA-treated group compared to the control ( P<0.05). Wound healing and Transwell assays demonstrated reduced migration and invasion capabilities in the experimental group relative to the control ( P<0.05). Compared to the model group, the LY2109761+ICA group exhibited further suppression of invasion ( P<0.05). Immunofluorescence revealed decreased Vimentin expression in the experimental group ( P<0.05), with an even more pronounced reduction in the LY2109761+ICA group ( P<0.01). Western blot analysis showed that the protein levels of N-cadherin, matrix metalloproteinase-9 (MMP9), Vimentin, TGF-β1, Smad2, and P-Smad2 were downregulated in the experimental group compared to the control ( P<0.05). These proteins were further suppressed in the LY2109761+ICA group compared to the model group ( P<0.05). ConclusionsICA inhibits TNBC cells proliferation, invasion, metastasis, and EMT by downregulating the TGF-β/Smad signaling pathway.