PD-L1 is expressed in tumor cells and its interaction with PD-1 plays an important role in evading immune surveillance; this can be overcome using PD-L1 or PD-1 immunotherapy antibodies. This study reports a novel approach for targeting PD-L1. In human breast cancer cell lines and 4T1 mouse mammary tumor cells PD-L1 expression was regulated by the nuclear receptor NR4A1/Sp1 complex bound to the proximal GC-rich region of the PD-L1 gene promoter. Treatment of breast cancer cells with bis-indole-derived NR4A1 antagonists including 1,1-bis(3'-indolyl)-1-(3-chloro-4-hydroxy-5-methoxyphenyl)methane (Cl-OCH3) decreased expression of PD-L1 mRNA, promoter-dependent luciferase activity and protein. In in vivo studies using a syngeneic mouse model bearing orthotopically injected 4T1 cells Cl-OCH3 decreased tumor growth and weight and inhibited lung metastasis. Cl-OCH3 also decreased expression of CD3+/CD4+/CD25+/FoxP3+ regulatory T cells and increased the Teff/Treg ratio. Therefore, the potent anti-cancer activities of NR4A1 antagonists are also accompanied by enhanced anti-tumor immunity in PD-L1-expressing triple-negative breast cancer and thus represent a novel class of drugs that mimic immunotherapy.