Galectin-3, which is a novel biomarker of cardiovascular stress and related to inflammation, could predict adverse cardiovascular events. However, its relationship with endothelial function in patients with chronic kidney disease (CKD) remains inconclusive. This study aimed to investigate the association between serum galectin-3 levels and endothelial function in patients with stages 3–5 CKD. Fasting blood samples were obtained from 130 patients. Serum galectin-3 levels were determined using the enzyme-linked immunosorbent assay. The endothelial function, demonstrated as a vascular reactivity index (VRI), was measured noninvasively through digital thermal monitoring test. Then, we sorted the patients into poor, intermediate, and good vascular reactivity (VRI < 1.0, 1.0 ≤ VRI < 2.0, and VRI ≥ 2.0), accounting for 24 (18.5%), 44 (33.8%), and 62 (47.7%) patients, respectively. As the VRI decreased, the serum galectin-3 and C-reactive protein (CRP) levels significantly increased. The galectin-3 value positively correlated with the CRP value but negatively correlated with estimated glomerular filtration rate. In multivariable stepwise linear regression analysis, serum log-transformed galectin-3 level and log-transformed CRP were significantly negatively associated with VRI values. Therefore, galectin-3 together with CRP is associated with VRI values and is a potential endothelial function modulator and a valuable biomarker of endothelial dysfunction in patients with CKD.