Tumor heterogeneity and interaction with tumor microenvironment play a crucial role in neoadjuvant chemotherapy (NAC) resistance in breast cancer (BRCA). Unraveling this dynamic interaction may help uncover novel therapeutic targets. Here, dynamic changes in tumor states and cellular composition are systemically characterized using 175,825 single-cell transcriptomics from na & iuml;ve and post-treatment biopsies of BRCA patients receiving NAC. CDK16+ tumors are identified featured with luminal progenitor cell (LPC)-like tumor cells enriched in the triple-negative subtype of BRCA, associated with chemo-resistance. Integrating single-cell RNA sequencing (scRNA-seq), spatial transcriptomics, and six independent public gene expression profiles that underwent chemotherapy revealed that POSTN+ cancer-associated fibroblasts (CAFs) are closely localized and interacted with CDK16+ LPC-like tumor cells to promote chemo-resistance. In vivo, CDK16 knockdown in tumor cells combined with chemotherapy significantly enhanced therapeutic efficacy. This in-house scRNA-seq from a mouse model validated that CDK16 knockdown reduced the LPC-like tumor cell signature, and the interaction of tumor featured with LPC-like tumor cells and POSTN+ CAFs. Together, the systematically integrated analyses uncovered an interaction network of CDK16+ tumor and POSTN+ CAFs that contributed to NAC- resistance, providing a new strategy for targeting CDK16 to enhance chemotherapy efficacy.