Background: Toluene is a common volatile organic compound (VOC) that usually enters the body through breathing or skin absorption. Blood urea nitrogen (BUN) is a by-product of protein metabolism and is commonly used as an indicator to assess renal function. alpha-Klotho protein is detected mainly in the kidney and has antioxidant, anti-aging, and nephroprotective properties. Objective: This study aimed to investigate the relationship between blood toluene concentration and BUN concentration and assess the alpha-Klotho protein's mediating role in this relationship. Methods: This study utilized data from the National Health and Nutrition Examination Survey (NHANES) from 2013 to 2016 on 2212 middle-aged and older Americans aged 40-79. Statistical methods such as multiple linear regression, threshold effects analysis, and mediation analysis were used to systematically assess the relationship between blood toluene concentration and BUN concentration and to explore the mediating role of alpha-Klotho protein. Results: Blood toluene concentration was significantly negatively associated with BUN concentration (p < 0.05). The results of the multiple regression analyses showed that the negative association remained statistically significant after controlling for confounding factors such as gender, age, race and body mass index. In addition, mediation analysis indicated that alpha-Klotho protein partially mediated this association (p < 0.05). Subgroup analyses showed a U-shaped relationship between blood toluene concentration and BUN concentration in lowincome populations. Conclusion: This study is the first to report a significant negative association between blood toluene concentration and BUN concentration in a middle-aged and elderly U.S. population, and suggests a potential partial mediating role of alpha-Klotho protein. These findings provide a new perspective for understanding how toluene affects renal function.