To assess the association between serum vitamin D levels and heart rate variability in patients with type 2 diabetes mellitus (T2DM). This study included 469 patients who were retrospective assessed for eligibility from Changzhou Second People’s Hospital, Affiliated Nanjing Medical University, between March 2013 and June 2020. A total of 191 T2DM patients were recruited and divided into 3 groups. A total of 191 patients were recruited. A significant difference was noted among groups for HbA1c (P < .001), serum uric acid (P = .048), and urea nitrogen (P = .043). The Vitamin D level in deficiency, insufficiency, and sufficient was 23.17, 38.89, and 63.01 nmol/L, respectively. The insufficient group had lower levels of percentage of normal-to-normal intervals differing by more than 50 milliseconds, and the square root of the mean of the squares of the differences between adjacent normal-to-normal R peak-to-R-peak time intervals than the sufficient vitamin D group. Furthermore, patients in deficiency and insufficiency group were associated with high level of low frequency power/high frequency power as compared with sufficient vitamin D group. Finally, serum 25-hydroxyvitamin D (25(OH)D) levels were positively correlated with rMSSD (P = .002). This study found that low serum 25(OH)D levels were associated with reduced heart rate variability parameters in patients with T2DM.