Background: The relationship between vitamin D and primary monosymptomatic nocturnal enuresis (PMNE) is still controversial. Objectives: This study aimed to investigate the relationship between different vitamin D levels with the development and severity of PMNE. Methods: A case-control study of 534 children (267 children with PMNE as the case group and 267 healthy children without enuresis as the control group), aged 6 - 15 years old, was conducted in two tertiary primary care hospitals, Tehran, Iran, from 2015 to 2021. The participants' demographic characteristics and vitamin D status were evaluated in both groups. The relationship between vitamin D levels with enuresis and its severity was studied as the main outcome measures. Multiple logistic regression models were utilized for statistical analyses to assay the odds ratio at a confidence interval of 95%. In this study, P < 0.05 were set as the significance level. Results: Vitamin D levels significantly differed between the two groups (18.58 ± 9.83 ng/mL and 30.23 ± 10.62 ng/mL, respectively; P < 0.001). After adjusting for confounders, vitamin D was significantly associated with enuresis (< 20 ng/mL, OR 3.07, 95% CI 1.9 - 4.95, P = 0.0001; 20 - 30 ng/mL, OR 2.72, 95% CI 1.47 - 3.67, P = 0.0001). Adjusted OR with 95% CI concerning the history of parental enuresis was 15.03 (95% CI 1.88 - 19.6, P = 0.01). The prevalence of vitamin D deficiency and insufficiency in the PMNE subjects were 107 cases (40.1%) and 109 cases (40.8%), respectively, and the prevalence values in the control group were 57 cases (21.3%) and 71 cases (26.6%), respectively (P < 0.05). Furthermore, the participants with higher frequencies of bedwetting represented lower vitamin D levels (severe, 10.6 ± 1.23 ng/ mL; moderate, 19.46 ± 1.21 ng/mL; mild, 26.8 ± 2.61 ng/mL, P = 0.0001). Conclusions: According to the finding of this study, parental history, and the participants’ vitamin D status were significant risk factors for the PMNE development. Vitamin D levels were significantly lower in the PMNE children. The prevalence of vitamin D deficiency and insufficiency was significantly higher in the enuretic subjects. Children with higher frequencies of bedwetting represented lower vitamin D levels.