The influence of Toll‑like receptor (TLR)4/myeloid differentiation factor (MyD)88 signaling on the invasion and metastasis of cancer cells has been previously reported. The purpose of the present study was to determine the role of TLR4/MyD88 in breast cancer cell migration and invasion, and to discover novel therapeutic targets for breast cancer treatment. TLR4, MyD88 and high mobility group box 1 (HMGB1) mRNA expression levels were assessed in highly invasive human MDA‑MB‑231 breast cancer cells, breast cancer cells with a low rate of invasion (MCF‑7) and normal human MDA‑Kb2 mammary gland cells by reverse transcription‑quantitative polymerase chain reaction. The protein expression levels of these markers were detected by western blotting and immunofluorescence. Randomly selected breast cancer and paracarcinoma tissues were used to measure TLR4 and MyD88 protein expression levels by immunohistochemistry. The mRNA and protein expression levels of TLR4 and MyD88 were significantly higher in MDA‑MB‑231 cells compared with either MCF‑7 cells or MDA‑Kb2 cells. The mRNA and protein expression levels of HMGB1 were comparable in the two breast cancer cell lines, with no statistical difference (P>0.05). TLR4 and MyD88 protein expression levels were also significantly higher in breast cancer tissues compared with paracarcinoma tissues (P<0.05). TLR4 and MyD88 protein expression levels were positively correlated with axillary lymph node metastasis and histological grade (P<0.05). TLR4/MyD88 expression levels were positively correlated with the metastasis of breast cancer cells. TLR4/MyD88 may be useful as a novel biomarker to evaluate the prognosis and treatment of patients with breast cancer.