Despite extensive basic and clinical research on immune checkpoint regulatory pathways, little is known about the effects of the ionic tumour microenvironment on immune checkpoint expression and function. Here, we describe a mechanistic link between Na+/K+ ATPase inhibition and activity of indoleamine-2′,3′-dioxygenase 1 (IDO1), a well-characterised immune checkpoint. IDO1 catalyses the rate-limiting step of tryptophan catabolism and inhibits the immune response to the tumour by local depletion of tryptophan, an amino acid essential for anabolic functions in cancer cells and T cells (Brochez, Chevolet and Kruse, 2017). To assess IDO1 function, we employed a medium-high throughput screening assay based on indirect spectrophotometric detection of kynurenine, a downstream tryptophan metabolite (Takikawa et al., 1988). Interferon-γ/tumour necrosis factor-α (IFN-γ/TNF-α) are used to stimulate kynurenine production in many human cell lines (Takikawa et al., 1988). Using this assay, we tested a library of 31 model ion channel targeting compounds for their effect on kynurenine production by IFN-γ/TNF-α stimulated breast cancer (MDA-MB-231) and lung cancer (A549) cells. We discovered that the cardiac glycosides ouabain and digoxin (Clark, Swanson and Stahl, 1975) inhibited kynurenine production in both MDA-MB-231 and A549 cancer cells. An initial screen of MDA-MB-231 resulted in decreased kynurenine (mean ± SD) upon treatment with 50 µM ouabain: 3.8 ± 1.3 µM, n=3, vs. 1% DMSO control: 27.5 ± 4.1 µM, n=6 (p<0.0001, one-way ANOVA Bonferroni post-hoc). An initial screen of A549 resulted in decreased kynurenine upon treatment with 50 µM ouabain: 1.5 ± 1.6 µM (conservatively, <3.1 µM), n=3, vs. 1% DMSO control: 15 ± 2.9 µM, n=6 (p<0.001, one-way ANOVA Bonferroni post-hoc). Titration of ouabain in the kynurenine assay resulted in half-maximal inhibitory concentrations (IC50s) of 89 nM for MDA-MB-231 and 17 nM for A549 cells. Titration of the related cardiac glycoside, digoxin, resulted in IC50s of 164 nM for MDA-MB-231 and 40 nM for A549 cells. Decreased cancer cell survival due to treatment with cardiac glycosides was measured by Alamar Blue absorbance (Abs570 nm – Abs600 nm) compared to 0.05% DMSO control (O’Brien et al., 2000). MDA-MB-231 cells decreased in number by 56% upon treatment with 500 nM ouabain and 33% upon treatment with 500 nM digoxin. A549 cells decreased in number by 39% upon treatment with 50 nM ouabain and 16% upon treatment with 50 nM digoxin. Thus, inhibition of kynurenine production may not be only due to cell death. Preliminary studies suggested that Na+/K+ ATPase inhibition by ouabain, but not digoxin, downregulated IDO1 expression in both cell lines (western blot, n=1), implying that the two inhibitors might alter tryptophan catabolism through distinct mechanisms. Ionic modulation via the Na+/K+ ATPase is a novel regulatory pathway of the immune checkpoint protein IDO1, with both mechanistic and clinical implications.