Studies have suggested that vitamin D deficiency is associated with impaired cardiac autonomic nerve function. This study explores the correlation between serum vitamin D and cardiac autonomic neuropathy (CAN) in prediabetes and reveals the protective effect of vitamin D against CAN. In total, 113 patients with prediabetes and definite CAN and 180 with prediabetes but without CAN were enrolled on the basis of their standard cardiovascular autonomic reflex test results. Chemiluminescence was used to measure 25(OH)D, the patients with CAN were divided into four groups, and the heart rate variability (HRV) of the study groups were compared. Relative to the 50≤25(OH)D<75-nmol/L group, the 25≤25(OH)D<50-nmol/L and 25(OH)D<25-nmol/L groups exhibited significant differences in the time and frequency domains of HRV (p< 0.05). Furthermore, we discovered that 25(OH)D is positively correlated with standard deviation of normal-to-normal RR intervals (SDNN) (β=0.566, p<0.05) and negatively correlated with low-to-high frequency ratio (LF/HF) (β=-0.199, p<0.05). A logistic regression reveals that CAN in prediabetes is significantly correlated with the 25(OH)D concentrations of <25 nmol/L (OR, 2.380 [1.208-4.691]; p<0.05) and 25≤25(OH)D<50 nmol/L (OR, 1.875 [1.064-3.751]; p<0.05). Serum 25(OH)D is significantly correlated with CAN in prediabetes, especially in the 25(OH)D <25-nmol/L group. Therefore, vitamin D deficiency may be related to the occurrence of CAN in prediabetes, and appropriate supplementation may provide protection against CAN.