Background Nocturnal enuresis (NE) is a common voiding problem in pediatric populations. Relatively, few studies have investigated the 25-Hydroxyvitamin D and NE associations in children, which may open up a new research area on the effect of vitamin D as nutritional therapy in the treatment of NE. The aim of this study was to determine the level of 25-hydroxyl vitamin D among children and adolescents with nocturnal enuresis in comparison to non-enuretic Egyptian children. Results Fifty children (24 females, 26 males) who presented with primary mono-symptomatic nocturnal enuresis (PMNE) and 50 healthy children (23 females, 27 males) were recruited in this study. A structured questionnaire focusing on demographic, socioeconomic, and frequency of bed-wetting were collected, and vitamin D serum levels were assessed in all children. In the NE group, the mean value of serum vitamin D levels was lower than the control (19.0 ± 6.5 versus 23.89 ± 4.19; P < 0.0001). Serum 25-hydroxyvitamin D levels were abnormal (< 20 ng/mL) in 46% of children with NE and 16% of controls (P < 0.001). The higher bed-wetting frequencies were associated with lower levels of 25(OH) D (Every night: 7.0 ± 1.4, 3–5 time/week: 11.2 ± 1.7, 1–3 times/week: 17.6 ± 3.7, < 2times/week: 22.4 ± 2.9, Once/6 month: 24.1 ± 4.3 (ng/ml), P < 0.001). Conclusion A difference in serum 25-hydroxyvitamin D levels was found between the two study groups. There is a need for more studies to explain vitamin D deficiency in larger series so that this test can be used in regular enuretic child investigations.