Breast cancer is one of the cancer-related leading causes of death worldwide. Treatment of breast cancer is complex and challenging especially when metastasis is developed. In this study, we established a new concept of using infrared radiation as an alternative approach to breast cancer treatment. We used middle infrared (MIR) in the wavelength range of 3 to 5 μm to irradiate breast cancer cells. MIR significantly inhibited cell proliferation in several breast cancer cells, but did not affect the growth of normal breast epithelium cells. We performed iTRAQ-coupled LC-MS/MS system to investigate the MIR-triggered molecular mechanisms in breast cancer cells. A total of 1,749 proteins were quantified and 167 proteins were considered to be regulated by MIR. Applying the functional enrichment analysis on the proteomics results, we confirmed that MIR caused G2/M cell cycle arrest, remodeled microtubule network to an astral pole arrangement, altered actin filament formation and focal adhesion molecule localization, and reduced cell migration activity and invasion ability. Together, our results uncover the collaborative effects of MIR regulated physiological responses in concentrated networks, demonstrating the potential implementation of infrared radiation in breast cancer therapy.