Breast cancer is the most common type of cancer among women. Chemotherapy is one of the main methods of breast cancer treatment, but this method is increasingly affected due to drug resistance. One of the newly discovered factors associated with drug resistance in cancer cells is interleukin receptor-associated kinase 1 (IRAK1). The aim of this study was to investigate the relationship between IRAK1 inhibition and sensitivity to methotrexate (MTX). Effects of various concentrations of MTX and constant concentration (1μg/ml) of IRAK1/4 inhibitor was examined on MCF-7, BT-20, BT-549, MB-468 cell lines. Cell viability was examined by water-soluble tetrazole -1, and cell apoptosis by flow cytometry. The expression of IRAK1 and BCRP genes was also assessed by the real-time PCR method. IRAK1 inhibitor decreased IC₅₀ in all examined cell lines, but the most prominent effect was observed in MB-468.  72 h incubation of cell lines with IRAK inhibitor and MTX, significantly increased the annexin-V and annexin-V/7AAD positive cells, suggesting an apoptotic effect of IRAK on all examined breast cancer cell lines. RT-qPCR test results showed that the IRAK inhibitor had no effect on the expression of BCRP at any time. Our results showed that the IRAK inhibitor can increase the chemosensitivity of breast cancer cell lines without effect on BCRP mRNA expression. IRAK inhibitor in combination with MTX can induce apoptosis in breast cancer cell lines.