Triple-negative breast cancer (TNBC) is a molecularly heterogeneous group of diseases defined by the lack of estrogen receptor (ER), progesterone receptor (PR) and absence of human epidermal growth factor receptor-2 (HER2) amplification. Consequently, TNBCs are impervious to therapies commonly used in other breast cancer subtypes and treatment options are largely limited to conventional genotoxic chemotherapy agents including doxorubicin. The long-term prognosis for TNBC patients with residual disease after chemotherapy is poor and a need exists to identify rational combination therapy approaches to improve the efficacy of chemotherapy for treating TNBC. Recent studies suggest that reprogramming of cellular metabolism is a component of the highly coordinated response to genotoxic stress. However, the metabolic response to clinically relevant genotoxic chemotherapy agents is poorly understood. We sought to identify adaptive metabolic reprogramming events triggered upon chemotherapy exposure that can be targeted to improve the efficacy of chemotherapy for treating TNBC. Using in vitro and in vivo metabolic profiling of TNBC cells, we have discovered that an increase in the abundance of pyrimidine nucleotides occurs in response to chemotherapy exposure. Mechanistically, the increase in pyrimidine nucleotides induced by chemotherapy is dependent on enhanced activity of the de novo pyrimidine synthesis pathway. We have found that pharmacological 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. Collectively, our studies provide pre-clinical evidence to demonstrate that adaptive reprograming of de novo pyrimidine synthesis represents a metabolic vulnerability that can be exploited to improve the anti-cancer activity of genotoxic chemotherapy agents for the treatment of TNBC.