Purpose <p>In patients with chronic kidney disease (CKD), vascular calcification (VC) is common and influences patient’s outcome and prognosis. However, evaluation methods for VC severity are limited. Klotho and fibroblast growth factor-23 (FGF-23) are biomarkers associating with VC development. This study aimed to explore the association of serum Klotho and FGF-23 levels with VC severity in patients with non-dialysis CKD.</p> Methods <p>Patients with non-dialysis CKD were enrolled during hospitalization and were divided into the following four groups on the basis of their coronary artery calcification (CAC) scores: non-VC (CAC scores = 0), mild VC (0 &lt; CAC scores ≤ 100), moderate VC (100 &lt; CAC scores ≤ 400), and severe VC groups (CAC scores &gt; 400). Serum Klotho and FGF-23 levels among the different groups were compared.</p> Results <p>A total of 154 non-dialysis CKD patients were enrolled. Correlation analysis showed that serum FGF-23 level (rho = 0.185, <i>p</i> = 0.022) was positively correlated with VC severity, whereas serum Klotho level (rho =  −  0.196, <i>p</i> = 0.015) was negatively correlated with VC severity in patients with CKD. Multivariable regression analysis showed that Klotho level [odd ratio (OR) = 0.998, 95% confidence interval (CI) 0.996–0.999, <i>p</i> = 0.001] served as a protective factor for VC severity in patients with CKD, whereas FGF-23 level (OR = 1.005, 95% CI 1.001–1.009, <i>p</i> = 0.020) was identified as risk factor for VC severity.</p> Conclusion <p>Serum Klotho and FGF-23 levels are potential predictors of VC severity in patients with non-dialysis CKD.</p>