Inosine (INO) enhances the potency of CAR-T cells by inducing stemness features and improving their functionality. Adenosine (Ado) suppresses immune responses in the tumor microenvironment, but INO, generated by adenosine deaminase (ADA), counteracts this by reprogramming metabolism and epigenetics. INO increases mitochondrial activity, glutaminolysis, and polyamine synthesis, promoting a stem-like state in CAR-T cells. Clinical-scale manufacturing using INO-supplemented media produced CAR-T cells with enhanced potency, effective in animal models. INO also reduces Ado-mediated suppression by increasing adenosine resistance. Transcriptomic and proteomic analyses show INO induces stemness-related gene expression and reprograms the epigenome. INO enhances CAR-T cell function, persistence, and antitumor activity, independent of glucose. INO modulates the epigenetic landscape, promoting memory-like T cells and enhancing immune responses. These findings suggest that INO is a potent modulator of CAR-T cell metabolism and epigenetic programming, offering a strategy to improve CAR-T cell potency for clinical applications.Inosine (INO) enhances the potency of CAR-T cells by inducing stemness features and improving their functionality. Adenosine (Ado) suppresses immune responses in the tumor microenvironment, but INO, generated by adenosine deaminase (ADA), counteracts this by reprogramming metabolism and epigenetics. INO increases mitochondrial activity, glutaminolysis, and polyamine synthesis, promoting a stem-like state in CAR-T cells. Clinical-scale manufacturing using INO-supplemented media produced CAR-T cells with enhanced potency, effective in animal models. INO also reduces Ado-mediated suppression by increasing adenosine resistance. Transcriptomic and proteomic analyses show INO induces stemness-related gene expression and reprograms the epigenome. INO enhances CAR-T cell function, persistence, and antitumor activity, independent of glucose. INO modulates the epigenetic landscape, promoting memory-like T cells and enhancing immune responses. These findings suggest that INO is a potent modulator of CAR-T cell metabolism and epigenetic programming, offering a strategy to improve CAR-T cell potency for clinical applications.