This study aimed to investigate the associations between serum α-Klotho levels and diabetic kidney disease (DKD), as well as all-cause mortality among individuals with diabetes. We included participants from the National Health and Nutrition Examination Survey (NHANES) between 2007 and 2016. Multivariable logistic regression and Cox proportional hazards models were used to evaluate the relationships of serum α-Klotho with DKD and mortality, respectively. Restricted cubic splines were applied to examine potential nonlinear associations. Subgroup analyses were performed to assess the robustness of the findings. A total of 3,098 participants were enrolled. The fully adjusted odds ratio (95% confidence interval (CI)) for the association between serum α-Klotho and DKD was 0.47 (0.33, 0.67; p < 0.001). Similarly, the hazard ratio for all-cause mortality was 0.61 (0.36, 0.96; p = 0.043). Restricted cubic spline analyses revealed nonlinear relationships between serum α-Klotho and both DKD and all-cause mortality. Kaplan-Meier curves indicated that participants in the lower quartiles of serum α-Klotho had significantly reduced survival probabilities. After Bonferroni correction, subgroup analyses showed no significant interactions between serum α-Klotho and all-cause mortality across populations. In conclusion, serum α-Klotho is significantly associated with DKD and all-cause mortality in diabetes.