Breast cancer is one of the most commonly diagnosed cancers in women, and due to the limitations of its therapeutic methods, new therapeutic method need to be developed. More and more evidences had shown the potential of low-temperature plasma (LTP) in cancer treatment. In this study, we investigated the inhibitory effect of LTP on the in vitro toxicity of breast cancer cells through the MTT assay, extracellular reactive oxygen species (ROS), and transcriptomics analysis. The results showed that under a certain treatment time, the cell viability was gradually decreased, and the extracellular ROS was gradually increased. In addition, the results of transcriptomics analysis showed that 1272 DEGs, 1573 DEGs, and 1272 DEGs were obtained in the 15s, 30s, and 45s treatment groups, respectively. DEGs were involved in MAPK signaling pathway, Fox O signaling pathway, Wnt signaling pathway, TGF-beta signaling pathway, chemical carcinogenesis-reactive oxygen species and mTOR Signaling pathway. In summary, our data suggested that LTP inhibited cell viability while inducing the activation of differentially expressed genes, representing a potential new therapy for breast cancer.