Objectives. The effects of Kluyveromyces marxianus on high-fat diet- (HFD-) induced kidney injury (KI) were explored. Methods. HFD-induced KI model was established using male C57BL/6 mice and treated with K. marxianus JLU-1016 and acid-resistant (AR) strain JLU-1016A. Glucose tolerance was evaluated via an oral glucose tolerance test (OGTT). KI was measured using Hematoxylin and Eosin (H&E) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis. The chemical indexes were analyzed, including lipid profiles, inflammatory cytokines, and creatinine. The levels of Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) or phospho-NF-κB p65 (Ser536) and alpha inhibitor of NF-κB (IκBα) were measured using qPCR and Western blot. The gut microbiota was sequenced using high-throughput sequencing. Results. HFD induction increased OGTT value, KI severity, oxidative stress, inflammatory cytokines, oxidative stress, apoptotic rate, creatinine levels, and the expression of TLR4/NF-κB, phospho-NF-κB p65 (Ser536), and IκBα deteriorated lipid profiles ( P < 0.05 ) and reduced gut microbiota abundance. K. marxianus treatment ameliorated HFD-induced metabolic disorders and reversed these parameters ( P < 0.05 ). Compared with the control, HFD induction increased the proportion of Firmicutes but reduced the proportion of Bacteroidetes and Lactobacillus. K. marxianus JLU-1016 and AR strain JLU-1016A treatments improved gut microbiota by reducing the proportion of Firmicutes and increasing the proportion of Bacteroidetes and Lactobacillus in the KI model ( P < 0.0001 ). Helicobacter has been identified with many infectious diseases and was increased after HFD induction and inhibited after K. marxianus JLU-1016 and AR strain JLU-1016A treatments. The strain JLU-1016A exhibited better results possibly with acid-tolerance properties to pass through an acidic environment of the stomach. Conclusions. K. marxianus may have a beneficial effect on KI by improving gut microbiota and inhibiting TLR4/NF-κB pathway activation.