Chimeric antigen receptor (CAR)-T therapy has shown superior efficacy against hematopoietic malignancies. However, many patients failed to achieve sustainable tumor control partially due to CAR-T cell exhaustion and limited persistence. In this study, by performing single-cell multi-omics data analysis on patient-derived CAR-T cells, we identify CD38 as a potential hallmark of exhausted CAR-T cells, which is positively correlated with exhaustion-related transcription factors and further confirmed with in vitro exhaustion models. Moreover, inhibiting CD38 activity reverses tonic signaling- or tumor antigen-induced exhaustion independent of single-chain variable fragment design or costimulatory domain, resulting in improved CAR-T cell cytotoxicity and antitumor response. Mechanistically, CD38 inhibition synergizes the downregulation of CD38-cADPR -Ca2+ signaling and activation of the CD38-NAD+-SIRT1 axis to suppress glycolysis. Collectively, our findings shed light on the role of CD38 in CAR-T cell exhaustion and suggest potential clinical applications of CD38 inhibition in enhancing the efficacy and persistence of CAR-T cell therapy.