INTRODUCTION: Diabetic kidney disease (DKD) represents a significant microvascular complication associated with diabetes and serves as a major contributor to end-stage renal disease. While many studies have highlighted the renal protective effects of the anti-aging protein Klotho, the potential link between Klotho and DKD within individuals with diabetes remains a subject of debate, and comprehensive studies utilizing large population-based databases are still needed. METHODS: This cross-sectional study, which is representative of the national population, examined data from US patients with diabetes aged 40-79 years, collected during the 2007-2016 cycles of the National Health and Nutrition Examination Survey (NHANES). Serum α-Klotho levels were determined using enzyme-linked immunosorbent assay (ELISA) techniques. Given that serum Klorho levels are not normally distributed, our analysis is based on values converted from the natural logarithm of Klotho. To assess the association between Klotho levels and the prevalence of DKD, multivariate regression models were utilized, taking into account potential confounding factors. Furthermore, we applied smooth curve fitting and segmented regression analyses to investigate possible threshold effects and identify inflection points. Subgroup analyses and cross-tests were performed to assess the consistency of associations in the general population. RESULTS: The investigation included 4490 individuals with diabetes, with an median age of 60.0 years and 48.2% of them being male. Among these participants, 1352 (30.1%) were diagnosed with DKD. The fully adjusted model (model 3) indicated a significant inverse relationship between Klotho levels and DKD prevalence. Statistical analysis showed that in fully adjusted model 3, each 1-unit increase in log-transformed Klotho was associated with a 42% reduction in DKD prevalence [OR 0.58, 95% CI (0.42, 0.80), p = 0.002]. Further analysis using smooth curve fitting revealed a U-shaped relationship between Klotho levels and DKD prevalence, with an inflection point at 6.82 (after natural logarithm conversion). CONCLUSIONS: This study identified a U-shaped relationship between Klotho levels and the prevalence of DKD in middle-aged and older adults with diabetes in the USA, with an inflection point of 6.82 (after natural logarithm conversion). Prior to this threshold, the relationship between Klotho and DKD prevalence was negatively correlated, while after the inflection point, the relationship became positive. Future studies are recommended to investigate the causal relationship behind this relationship.