Abstract: Background: Vitamin D deficiency has reached pandemic levels in the Middle East and North Africa (MENA) region, even though sunshine is abundant all year round for the cutaneous synthesis of vitamin D through the skin. This study aimed to determine the prevalence of vitamin D deficiency and risk factors associated with serum 25-hydroxy vitamin D (25(OH)D) in children and adolescents aged from 10 to 19 years, as well as the possible associations of vitamin D with calcium, magnesium and phosphate levels. Methods: A multi-center, cross-sectional study was conducted between May and August 2019 at the Ministry of Health in the Kingdom of Bahrain. A total of 383 boys and girls were selected from five health centers from five different regions in the Kingdom of Bahrain. Information about sex, age, education level, weight, height, degree of sunlight exposure, and physical activity levels was recorded. A blood sample was taken from each participant to test serum levels of 25(OH)D, calcium, magnesium and phosphate. Results: The results revealed that 92.1% of the participants were deficient in vitamin D. A significantly higher percentage of boys (96.2%) were vitamin D deficient (<20 ng/mL) than girls (88.3%) (p value = 0.004). Vitamin D deficiency were more prevalent among overweight (96.8%) and obese (96.2%) participants than normal body weight and wasted participants (p value < 0.001). Being male, overweight, or obese was significantly positively associated with a risk of vitamin D deficiency. Vitamin D deficiency was significantly associated with low serum levels of magnesium. No significant associations were detected between vitamin D deficiency and calcium and phosphate serum levels. However, vitamin D deficiency was significantly associated with low serum level of magnesium (p value = 0.017). Conclusions: Our study revealed that vitamin D deficiency was more prevalent among overweight and obese adolescents and mostly boys rather than girls. Magnesium and phosphate were lower in adolescents and children with lower serum 25(OH)D, showing a clear association between these biomarkers and the 25(OH)D.