To investigate the relationship between the gut microbiota and the biological characteristics of patients with newly diagnosed diffuse large B-cell lymphoma (DLBCL).This prospective study included 71 patients with newly diagnosed DLBCL. The microbiota was analyzed using 16S ribosomal DNA sequencing of fecal samples. Blood cytokines, PD-1, and PD-L1 were measured by enzyme-linked immunosorbent assay. Stratified analyses based on clinical characteristics were conducted to investigate correlations between alterations in gut microbiota and key clinicopathological parameters of DLBCL.In the analysis of gut microbiota heterogeneity (α diversity index), species abundance was significantly higher in the International Prognostic Index (IPI) < 3 group compared to the IPI ≥ 3 group (high-risk group). Ruminococcus was increased in the IPI ≥ 3 group. Parabacteroides, Ruminococcus, and Eubacterium were increased in the non-germinal center B-cell-like group, while Lachnospira was decreased. Megamonas was significantly increased in the high β2-microglobulin group, while Lactobacillus reuteri and Lachnospira were decreased. In the low CD4+/CD8+ ratio group, Parabacteroides was increased, while Akkermansia was decreased. Patients who failed to achieve complete remission at interim evaluation showed marked increases in Ruminococcus and Alistipes. Extranodal involvement was associated with elevated Alistipes and Enterococcus.This study identified a potential relationship between the gut microbiota and DLBCL characteristics, highlighting specific gut microbiota organisms that may influence disease development and progression.AJCR Copyright © 2025.