Chemotherapy resistance is a major barrier to the treatment of triple-negative breast cancer (TNBC), and strategies to circumvent resistance are required. Using in vitro and in vivo metabolic profiling of TNBC cells, we show that an increase in the abundance of pyrimidine nucleotides occurs in response to chemotherapy exposure. Mechanistically, elevation of pyrimidine nucleotides induced by chemotherapy is dependent on increased activity of the de novo pyrimidine synthesis pathway. Pharmacologic inhibition of de novo pyrimidine synthesis sensitizes TNBC cells to genotoxic chemotherapy agents by exacerbating DNA damage. Moreover, combined treatment with doxorubicin and leflunomide, a clinically approved inhibitor of the de novo pyrimidine synthesis pathway, induces regression of TNBC xenografts. Thus, the increase in pyrimidine nucleotide levels observed following chemotherapy exposure represents a metabolic vulnerability that can be exploited to enhance the efficacy of chemotherapy for the treatment of TNBC.Significance: The prognosis for patients with TNBC with residual disease after chemotherapy is poor. We find that chemotherapy agents induce adaptive reprogramming of de novo pyrimidine synthesis and show that this response can be exploited pharmacologically, using clinically approved inhibitors of de novo pyrimidine synthesis, to sensitize TNBC cells to chemotherapy. Cancer Discov; 7(4); 391-9. ©2017 AACR.See related article by Mathur et al., p. 380This article is highlighted in the In This Issue feature, p. 339.