Previous studies have demonstrated that reduced heart rate variability (HRV) and hypovitaminosis D are associated with cardiovascular disease (CVD). However, few reports have investigated the effects of vitamin D on HRV. This cross-sectional study analyzed serum 25-hydroxyvitamin D (25(OH)D) and HRV indices using 5-min R-R interval recordings with an automatic three-channel electrocardiography in healthy subjects (103 males and 73 females). Standard deviation of N-N interval (SDNN), square root of mean squared differences of successive N-N intervals (RMSSD), total power (TP), very low frequency (VLF), low frequency (LF), and high frequency (HF) were reported. The mean age of subjects was 55.3 ± 11.3 years and the mean 25(OH)D level was 21.2 ± 9.9 ng/mL. In a multiple linear regression model, 25(OH)D was positively correlated with SDNN (β = 0.240, p < 0.002), and LF (β = 0.144, p = 0.044). Vitamin D deficiency (25(OH)D < 15 ng/mL) was associated with decreased SDNN (<30 m/s) (OR, 3.07; 95% confidence interval (CI), 1.32-7.14; p = 0.014) after adjusting for covariates. We found that lower 25(OH)D levels were associated with lower HRV, suggesting a possible explanation for the higher risk of CVD in populations with hypovitaminosis D.