Indoleamine 2,3 dioxygenase (IDO) is upregulated in many tumor types, including breast cancer, and plays a reputable role in promoting tumor immune tolerance. The importance of the immunosuppressive mechanism of IDO by suppressing T-cell function has garnered profound interest in the development of clinical IDO inhibitors. Herein, we established a screening method with cervical HeLa cells to induce IDO expression using interferon-γ (IFN-γ). After screening our chemical library, we found that salinomycin potently inhibited IFN-γ-stimulated kynurenine synthesis with IC50 values of 3.36-4.66 μM in both human cervical and breast cancer cells. Salinomycin lowered the IDO1 and IDO2 expression with no impact on the expression of tryptophan-2,3-dioxygenase. Interestingly, salinomycin potently repressed the IDO1 enzymatic activity by directly targeting the proteins in cells. Molecular docking revealed an alignment that favors nucleophilic attack of salinomycin in the catalytic domain of IDO1. Activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway by IFN-γ was significantly suppressed by salinomycin, via inhibiting the Jak1, Jak2, and STAT1/3 phosphorylation. Moreover, it inhibited IFN-γ-induced activation of the nuclear factor (NF)-κB pathway by inhibiting IκB degradation and NF-κB phosphorylation without affecting BIN1 expression. Furthermore, salinomycin significantly restored the proliferation of T cells co-cultured with IFN-γ-treated breast cancer cells and potentiated antitumor activity of cisplatin in vivo. These findings suggest that salinomycin suppresses kynurenine synthesis by inhibiting the catalytic activity of IDO1 and its expression by inhibiting the JAK/STAT and NF-κB pathways. Salinomycin warrants further investigation as a novel dual-functional IDO inhibitor for cancer immunotherapy. Copyright © 2020 Elsevier Inc. All rights reserved.