Triple negative breast cancer (TNBC) is associated with epithelial-mesenchymal transition (EMT) and an enrichment in cancer stem cells (CSC) which are both involved in tumor chemoresistance and metastasis. Our group has shown that RUNX1 is implicated in the aggressiveness of this breast cancer subtype by promoting cell migration and regulating tumor gene expression, and in chemoresistance of TNBC-androgen responder cell lines. But the mechanisms involved are still undetermined. Moreover, RUNX1 protein expression in TNBC correlates with poor patient prognosis. Our aim was to evaluate RUNX1 relevance during drug treatment in human TNBC. Here we show that using a RUNX1 transcriptional activity commercial inhibitor (AI-10-104) in TNBC MDA-MB-231 and -468 cell lines there is a significant decrease in cell viability and migration and a significant increase in apoptosis. Interestingly, in a forced suspension cell model (which promotes a CSC phenotype), RUNX1 expression is significantly increased compared to the attached cell population, and the inhibition of RUNX1 transcriptional activity decreases OCT4, ABCC1 and ALDH1 gene expression in the forced-suspended subpopulation. Moreover, RUNX1 inhibition in the MDA-MB-231 cell line prevents mammosphere formation capacity. RUNX1 mRNA is significantly upregulated in doxorubicin (Doxo)- and paclitaxel (Px)-treated cell lines. Also, its protein expression is upregulated in PX resistant cell lines. We determined that loss of RUNX1 transcriptional activity significantly enhances Doxo and Px toxicity in TNBC cell lines by reducing viability and enhancing apoptosis. Also, mammospheres already established and then treated simultaneously with Doxo and AI-10-104 show a significant number reduction compared with Doxo alone. In addition, PX-resistant MDA-MB-468 cell line treated with AI-10-104 recovers Px sensitivity. Therefore, our data strongly suggests that RUNX1 may be involved in the generation of TNBC chemoresistant cells.