Lapatinib is highly active against breast cancer with HER2 overexpression in preclinical and clinical settings. Constitutive activation of NF-κB is linked to proliferation and apoptosis in breast cancer cells. NF-κB can be activated by HER2 in breast cancer cells. However, the effect of lapatinib on NF-κB activity is not completely clear. In this study, we showed that lapatinib potently inhibited activation of NF-κB in HER2-overexpressing breast cancer cells, including SKBR3 and MDA-MB-453; but not in non-HER2-overexpressing breast cancer cells, MDA-MB-231, MDA-MB-468 and MDA-MB‑435. In addition, we established a model of acquired resistance to lapatinib by chronically challenging SKBR3 breast cancer cells with increasing concentrations of lapatinib. EMSA assays showed that there was decreased NF-κB activity in the resistant cells. Western blot analysis showed that lapatinib reduced the phosphorylation of IκB-α in a time- and dose-dependent manner in SKBR3 cells. Furthermore, the expression level of p-IκB-α protein was markedly decreased in the resistant cells, compared with the parental SKBR3 cells. Additionally, treatment with the PI3K inhibitor LY294002 dramatically inhibited activation of NF-κB in HER2-overexpressing breast cancer cells. Moreover, LY294002 inhibited phosphorylation of Akt and IκB-α in SKBR3 cells. Our results suggest that lapatinib potently inhibits the activation of NF-κB in HER2-overexpressing breast cancer cells. Lapatinib appears to inactivate NF-κB through reducing phosphorylation of IκB-α via blocking the PI3K/Akt cascade.