Aim To investigate the relationship between serum levels of fibroblast growth factor 23 (FGF-23), Klotho protein and carotid atherosclerosis in non-dialysis patients with chronic kidney disease (CKD) 2-5 stage. Methods 126 CKD 2-5 stage patients were involved in the study. Carotid intima-media thickness (CIMT) was measured by color Doppler ultrasonography. The patients were divided into CIMT thickening group and CIMT normal group according to CIMT. Serum levels of FGF-23 and Klotho protein were determined by enzyme-linked immunosorbent assay, and the general data and clinical biochemical indexes were collected. The relative indexes of the two groups were compared, and the correlation between FGF-23, Klotho protein and carotid atherosclerosis was analyzed. Unconditional Logistic regression analysis was used for multivariate analysis of CIMT influencing factors. Results The serum FGF-23 level in CIMT thickening group was significantly higher than that in CIMT normal group (435.39±221.20 vs 360.22±194.26, P＜0.05), and the level of Klotho protein was significantly lower than that in CIMT normal group (446.54±132.49 vs 499.36±121.38, P＜0.05). Multivariate stepwise regression analysis showed that Klotho protein was an independent protective factor for CIMT thickening in patients with CKD (OR＝1.086, P＜0.05), while age increase and FGF-23 were independent risk factors for CIMT thickening in patients with CKD (OR＝1.075, P＜0.05; OR＝1.238, P＜0.05). Conclusion CIMT thickening is related to FGF-23 and Klotho proteins in non-dialysis patients with CKD 2-5 stage. FGF-23 and Klotho protein play an important role in the occurrence and development of carotid atherosclerosis.