Metabolic alterations are closely associated with the exhaustion and immune deficiency of CD8+ tumor-infiltrating lymphocytes (TILs), while little is known about diffuse large B-cell lymphoma (DLBCL). This study aimed to elucidate the significance of the metabolic alterations in exhausted CD8+TILs and its underlying regulatory mechanism in DLBCL.The metabolic alterations in exhausted CD8+TILs in DLBCL were evaluated through single-cell RNA sequencing (scRNA-seq). The crucial metabolic pathway and its significance in the biological function of exhausted CD8+TILs were investigated by scRNA-seq and RNA sequencing. The marker gene in crucial metabolic pathway, and its correlations between exhaustion status, the tumor microenvironment (TME) composition, clinicopathological characteristics, prognosis, and immune checkpoint blockade (ICB) therapy efficacy were evaluated by scRNA-seq, RNA sequencing, immunohistochemistry, and RT-qPCR. Furthermore, the underlying regulatory mechanism involved in the metabolic alteration related to CD8+TILs exhaustion was explored through scRNA-seq, RNA sequencing, and somatic mutation analysis.Our study illustrated the metabolic heterogeneity in CD8+TILs, and demonstrated that oxidative phosphorylation (OXPHOS) was the crucial pathway in CD8+TILs exhaustion. The high OXPHOS activity indicated the immune deficiency in exhausted CD8+TILs, and UQCRFS1 was identified as a marker gene. High UQCRFS1 indicated the immunosuppressive TME, severe clinicopathological characteristics, including activated B-cell-like subtype, high IPI and PS score, advanced stage, dismal prognosis, and resistance to ICB therapy. Furthermore, MYC-related signaling and P2RY8 mutation in DLBCL may regulate the UQCRFS1 expression in exhausted CD8+TILs.Our study highlights the importance of OXPHOS activity in CD8+TILs exhaustion and suggests its possible regulatory mechanism, which is feasible in clinical evaluation and beneficial for novel immunotherapeutic approaches in DLBCL.© 2025. The Author(s).