Despite extensive basic and clinical research on immune checkpoint regulatory pathways, little is known about the effects of the ionic tumor microenvironment on immune checkpoint expression and function. Here we describe a mechanistic link between Na+/K+-ATPase (NKA) inhibition and activity of the immune checkpoint protein indoleamine-pyrrole 2',3'-dioxygenase 1 (IDO1). We found that IDO1 was necessary and sufficient for production of kynurenine, a downstream tryptophan metabolite, in cancer cells. Based on this, we developed a spectrophotometric assay to screen a library of 31 model ion transport-targeting compounds for potential effects on IDO1 function in A549 lung and MDA-MB-231 breast cancer cells. This screen revealed that the cardiac glycosides ouabain and digoxin inhibited kynurenine production at concentrations that did not affect cell survival. Furthermore, NKA inhibition by ouabain and digoxin resulted in increased intracellular Na+ levels and downregulation of IDO1 mRNA and protein levels, which was consistent with the reduction in kynurenine levels. Additionally, knockdown of ATP1A1, the ɑ1 subunit of the NKA and target of cardiac glycosides, increased Na+ levels to a lesser extent than cardiac glycoside treatment, and did not affect IDO1 expression. However, ATP1A1 knockdown significantly enhanced the effect of cardiac glycosides on IDO1 expression and kynurenine production. Mechanistically, we show that cardiac glycoside treatment resulted in curtailing the length of phosphorylation-mediated stabilization of STAT1, a transcriptional regulator of IDO1 expression, an effect enhanced by ATP1A1 knockdown. Overall, our findings reveal cross-talk between ionic modulation via cardiac glycosides and immune checkpoint protein expression in cancer cells with broad mechanistic and clinical implications.