Background: Cross talk of tumor–immune cells at the gene expression level has been an area of intense research. However, it is largely unknown at the alternative splicing level which has been found to play important roles in the tumor–immune microenvironment. Results: Here, we re-exploited one transcriptomic dataset to gain insight into tumor–immune interactions from the point of AS level. Our results showed that the AS profiles of triple-negative breast cancer cells co-cultured with activated T cells were significantly changed but not Estrogen receptor positive cells. We further suggested that the alteration in AS profiles in triple-negative breast cancer cells was largely caused by activated T cells rather than paracrine factors from activated T cells. Biological pathway analyses showed that translation initiation and tRNA aminoacylation pathways were most disturbed with T cell treatment. We also established an approach largely based on the AS factor–AS events associations and identified LSM7, an alternative splicing factor, may be responsible for the major altered events. Conclusions: Our study reveals the notable differences of response to T cells among breast cancer types which may facilitate the development or improvement of tumor immunotherapy. How to cite: Zhao L, Yang X, Feng C, et al. Triple-negative breast cancer cells respond to T cells severely at the alternative splicing layer. Electron J Biotechnol 2021;50.https://doi.org/10.1016/j.ejbt.2021.01.001 © 2021