Abstract： Objective:To investigate the effect of eicosapentaenoic acid (EPA) on pyrolysis of HER2-positive breast cancer cells.Methods:EPA was used to treat HER2-positive breast cancer cells prior to detection of EPA cytotoxicity on the HER2-positive breast cancer cells. Annexin V and propidium iodide (Annexin V/PI) assay was used to determine whether the EPA-reduced cell viability is associated with increased cell death. After EPA treatment, the expression of major molecular markers in the cell pyrolytic pathway was examined.Results:After EPA treatment, there was a reduction in viability of HER2-positive breast cancer cell-lines AU-565, SK-BR-3, and MDA-MD-435 ( P<0.05) , but not significantly in normal breast cell-liness MCF 10A and Hs 578Bst ( P>0.05) . After EPA treatment for 24 h, the cell-lines AU-565, SK-BR-3, and MDA-MD-435 cells increased in Annexin+/PI+ cell counts ( P<0.05) , while the cell-lines MCF 10A and Hs 578Bst increased in Annexin+/PI- cell counts ( P<0.05) . After EPA treatment, the phosphorylation of the major proteins p65, IKKα/β, and IκBα in the NF-κB pathway increased in cell-lines AU-565, SK-BR-3, and MDA-MD-435, but not significantly in cell-lines MCF 10A and Hs 578Bst. In addition, molecular docking study revealed that EPA may interact with NEMO (at the K381) , an upstream protein of the NF-κB pathway, with the lowest binding energy being -7.33 kcal/mol. After EPA treatment, the annexin+/PI+ cell counts in NEMO knockout MDA-MD-435 cells were significantly reduced ( P<0.05) . Overexpression of wild-type or K381R-mutated NEMO in NEMO knockout cells showed that, the Annexin+/PI+ cell counts were significantly reduced in MDA-MD-435 cells with K381R-mutated NEMO overexpression ( P<0.05) . After EPA treatment, the AU-565, SK-BR-3 and MDA-MD-435 cells showed lowered expression of Pro-IL-1β and ASC, increased expression of cleaved caspase 1 and cleaved Gasdermin D, higher secretion of IL-1β ( P<0.05) , and cytoplasm translocation of HMGB1; the MCF 10A and Hs 578Bst cells did not show significant change in expression of Pro-IL-1β, ASC, cleaved caspase1, and cleaved Gasdermin D, IL-1β secretion ( P>0.05) , or cytoplasm translocation of HMGB1. Conclusion:EPA interacts with NEMO at K381 domain, thereby activates the NF-κB pathway, promotes the cleavage of caspase1, IL-1β and Gasdermin D, increases the IL-1β secretion and nuclear translocation of HMGB1, and ultimately induces pyroptosis of HER2 positive breast cancer cells.