NF-κB transcription factor has been associated with cancer development and chemoresistance. We studied the signaling pathway activated by doxorubicin (DOX) leading to NF-κB activation in breast cancer cells. NF-κB activity was evaluated by electrophoretic mobility shift in T47D, ZR75.30 and primary culture (MBCDF) from a ductal infiltrating carcinoma. Cell viability was measured by crystal violet. Western blotting was performed to check the expression and phosphorylation of IκBα Ser-32/36. c-Abl was inhibited with Imatinib or by overexpressing a dominant negative form of c-Abl (K290R). We found a correlation between sensitivity to DOX and amplitude of NF-κB activation. In cells least sensitive to DOX, NF-κB remained activated for longer time (T47D and MBCDF). The opposite effect was observed in cells sensitive to DOX (ZR75.30). DOX did not induce IκBα degradation or Ser-32/36 phosphorylation. Instead, there were modifications in the levels of IκBα tyrosine phosphorylation, suggesting an atypical NF-κB activation. In DOX-resistant cells, Imatinib treatment reduced IκBα tyrosine phosphorylation and NF-κB activity. The Imatinib–DOX combination significantly enhanced cell death of T47D and MBCDF breast cancer cells. Overexpression of c-Abl K290R in T47D and MBCDF cells reduced basal and DOX-induced NF-κB activation as well as IκBα tyrosine phosphorylation. In c-Abl K290R cells, DOX treatment did not mimic the combination Imatinib–DOX-induced cell death. Inhibition of c-Abl inactivated IκBα/NF-κB pathway is associated with IκBα tyrosine phosphorylation in breast cancer cells. These results also raise the potential use of a combined therapy with Imatinib and DOX for breast cancer patients.