Lipopolysaccharide (LPS), a potent endotoxin present in the outer membrane of Gram‑negative bacteria, causes chronic immune responses associated with inflammation. In the present study, the association between LPS and the dysbiosis of Gram‑negative bacteria in the gut microbiome was determined in patients with type 2 diabetes mellitus (T2DM) and chronic kidney disease (T2DM‑CKD; stages 4 and 5, not on dialysis) compared with healthy individuals. Microbiome diversity was analyzed in patients with T2DM‑CKD and healthy controls by sequencing the hypervariable sub‑regions of the 16S ribosomal RNA gene from stool samples. Serum samples were assayed by ELISA for LPS, C‑reactive protein (CRP), tumor necrosis factor‑α (TNFα), interleukin‑6 (IL6) and endothelin‑1. A total of four gut Gram‑negative phyla (Bacteroidetes, Proteobacteria, Fusobacteria and Verrucomicrobia) were identified in the gut microbiome of the T2DM‑CKD and control groups. Proteobacteria, Verrucomicrobia and Fusobacteria exhibited significantly increased relative abundance in patients with T2DM‑CKD when compared with controls (P< 0.05). The levels of serum LPS were significantly increased in patients with T2DM‑CKD compared with controls (P< 0.05). Elevated serum LPS was significantly correlated with increased levels of TNFα, IL6 and CRP. The dysbiosis of Gram‑negative bacteria in the gut microbiome of patients with T2DM‑CKD may contribute to the elevated serum levels of LPS and the consequential effects on the inflammatory biomarkers in these patients. The association between the dysbiosis of Gram‑negative bacteria in the gut microbiome of patients with T2DM‑CKD, increased LPS levels and the effects on inflammatory biomarkers may provide insight into potential diagnostic and therapeutic approaches in the treatment of T2DM‑CKD.