Abstract Background Immune infiltration of breast cancer is associated with clinical outcome. A growing number of research suggests the immune response were composed by variously functionally distinct immune cell. But it’s not clear which kinds of cell play the important role. The aim of this study was to determine whether differences in the cellular composition of the immune infiltrate in breast cancer influence survival and treatment response, and construct a stromal immunotype which could predict the response of neoadjuvant therapy and survival. Patients and Methods A total of 1502 ER negative breast cancers from TCGA and METABRIC cohort were used to infer the proportions of 22 subsets of immune cells, Another 320 ER negative breast cancer patients from Guangdong Provincial People’s Hospital in the validation cohort were also included in the study. Immune cell infiltration was evaluated by immunohistochemical staining or CIBERSORT method, Five immune features were selected out of 22 immune features to construct immunotype based on LASSO Cox regression model. ResultOf the cell subsets investigated, CD8+ T cells (hazard ratio [HR] = 0.064, 95% CI 0.018-0.234; p < 0.001), CD4+ T cells (HR 0.072, 95% CI 0.013-0.382; p = 0.002), B cells (HR 0.04, 95% CI 0.005-0.340; p = 0.003) , M1 macrophages(HR 0.09, 95% CI 0.016-0.502; p = 0.006) were associated with favourable outcome. T regulatory cells(HR 10.791, 95% CI 2.059-58.444; p = 0.005) emerged as the most strongly associated with poor outcome. Using the LASSO model, we classified ER negative breast cancer patients into stromal immunotype A subgroup (CD8+T cellshigh CD4+T cellshigh B cellhigh M1 macrophageshigh Treglow) and stromal immunotype B subgroup (CD8+T cellslow CD4+T cellslow B celllow M1 macrophageslow Treg high). Significant differences were found between immunotype A and immunotype B in the combined cohort with 10-year overall survival (66.2% vs. 49.8%; P<0.001) and 10-year disease-free survival (63.8% vs. 44.4%; P<0.001). Stromal immunotype was revealed to be an independent prognostic indicator in multivariate analysis in all cohorts separately, and also showed to be related to pCR in neoadjuvant chemotherapy. Finally, stromal immunotype A showed higher immune checkpoint molecules (PD-L1, PD-1, CTLA-4) expression and three important cytokines expression profiles (IL-2, INF-γ and TGF-β). Conclusion The stromal immunotypes could predict survival and recurrence of ER negative breast cancer patients effectively. Furthermore, the immunotypes might be a practical predictive tool for immunotherapy and neoadjuvant chemotherapy. Citation Format: Fei Ji, Ciqiu Yang, Liulu Zhang, Mei Yang, Jieqing Li, Hongfei Gao, Teng Zhu, Minyi Cheng, Kun Wang. Identification and validation of stromal immunotype predict survival and benefit from neoadjuvant chemotherapy in patients of breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-11.