Adenosine (Ado) is a potent immunosuppressant in the tumor microenvironment, and exhausted CD8+ CAR-T cells express CD39 and CD73, which mediate Ado generation. To enhance CAR-T cell potency, researchers explored knocking out CD39, CD73, or adenosine receptor 2a (A2aR), but observed only modest effects. Instead, overexpression of adenosine deaminase (ADA) or exposure to inosine (INO), the代谢产物 of ADA, significantly enhanced CAR-T functionality and induced stemness features. INO reprogrammed the metabolic profile of CAR-T cells, reducing glycolysis and increasing glutaminolysis, polyamine synthesis, and mitochondrial activity. This metabolic reprogramming was associated with epigenetic changes, leading to greater stemness. Clinical-scale manufacturing using INO-generated CAR-T cells showed enhanced potency and met clinical dosing criteria. These findings identify INO as a potent modulator of CAR-T cell metabolism and epigenetic programming, providing an enhanced potency platform for cell manufacturing.Adenosine (Ado) is a potent immunosuppressant in the tumor microenvironment, and exhausted CD8+ CAR-T cells express CD39 and CD73, which mediate Ado generation. To enhance CAR-T cell potency, researchers explored knocking out CD39, CD73, or adenosine receptor 2a (A2aR), but observed only modest effects. Instead, overexpression of adenosine deaminase (ADA) or exposure to inosine (INO), the代谢产物 of ADA, significantly enhanced CAR-T functionality and induced stemness features. INO reprogrammed the metabolic profile of CAR-T cells, reducing glycolysis and increasing glutaminolysis, polyamine synthesis, and mitochondrial activity. This metabolic reprogramming was associated with epigenetic changes, leading to greater stemness. Clinical-scale manufacturing using INO-generated CAR-T cells showed enhanced potency and met clinical dosing criteria. These findings identify INO as a potent modulator of CAR-T cell metabolism and epigenetic programming, providing an enhanced potency platform for cell manufacturing.