Serum vitamin B12 and α-Klotho are important markers associated with aging. Limited studies have been conducted on the relationship between vitamin B12 and α-Klotho. This study investigated the relationship between circulating α-Klotho and vitamin B12. A total of 4,502 American adults with circulating vitamin B12 levels and α-Klotho levels from the National Health and Nutrition Examination Survey (2011-2014) were included. A weighted multiple linear regression model was used to evaluate the correlation between vitamin B12 and α-Klotho levels. To clarify potential non-linearities, smoothed curve fitting and threshold effects analysis were employed. A statistically significant non-linear relationship was found between vitamin B12 levels and circulating α-Klotho levels after adjusting for potential confounders. We observed an inverted U-shaped relationship between serum vitamin B12 levels and circulating α-Klotho levels. Notably, serum vitamin B12 levels below the threshold (1,020 pg/mL) exhibited a positive correlation with circulating α-Klotho levels (β = 0.14, 95% confidence interval (CI): 0.09-0.18, p < 0.0001). Conversely, serum vitamin B12 levels above the threshold (1,020 pg/mL) exhibited a negative correlation with circulating α-Klotho levels (β = -0.12,95% CI: -0.17--0.06, p < 0.0001). Sensitivity analyses were performed and consistent results were obtained. This study demonstrated an inverted U-shaped relationship between circulating vitamin B12 and α-Klotho in American adults. The optimal concentration of serum vitamin B12 in American adults was found.