Enhanced infiltration of regulatory T (Treg) cells into tumor tissue is detrimental to cancer patients and closely associated with poor prognosis as they create an immunosuppressive state that suppresses anti-tumor immune responses. Therefore, breaking Treg-mediated immune tolerance is important when considering cancer immunotherapy. Here we show that the Nr4a nuclear receptors, key transcription factors maintaining Treg cell genetic programs, contribute to Treg-mediated suppression of anti-tumor immunity in the tumor microenvironment. Mice lacking Nr4a1 and Nr4a2 genes specifically in Tregs showed resistance to tumor growth in transplantation models without exhibiting any severe systemic autoimmunity. The chemotherapeutic agent camptothecin (CPT) and a common cyclooxygenase-2 (COX-2) inhibitor were found to inhibit transcriptional activity and induction of Nr4a factors, and they synergistically exerted anti-tumor effects. Genetic inactivation or pharmacological inhibition of Nr4a factors unleashed effector activities of CD8+ cytotoxic T cells and evoked potent anti-tumor immune responses. These findings demonstrate that inactivation of Nr4a in Tregs breaks immune tolerance toward cancer, and pharmacological modulation of Nr4a activity may be a novel cancer treatment strategy targeting the immunosuppressive tumor microenvironment.