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Purpose: Sleep duration is associated with aging. However, the relationship between sleep duration and the concentration of the protein klotho in the serum remains unknown in the general population of the United States. Hence, this study aimed at exploring the association between them. Methods: Participants whose data included klotho protein and sleep duration variables in the National Health and Nutrition Examination Survey data from 2007 to 2016 were utilized for this analysis. Results: Sleep duration was non-linearly associated with the level of klotho protein in the serum, with a negative association between sleep duration and serum klotho concentration after adjusting for confounding variables (β = − 7.6; 95% CI: − 11.3, − 4.0; P < 0.001). The conversion of the sleep duration from a continuous variable to a categorical variable (tertile: T1: < 5.5 hours; T2: 5.5– 7.5 hours; T3: > 7.5 hours) revealed that the serum klotho of the participants in the highest tertile (> 7.5 hours) was 21.9 pg/mL lower (95% CI: − 38.6, − 5.2; P = 0.01) than those in the lowest tertile (< 5.5 hours). Conclusion: Our results revealed that people who sleep more than 7.5 hours per night have decreased levels of the anti-aging protein klotho in their serum, thus being more at risk of aging-related syndromes. [ABSTRACT FROM AUTHOR]