Introduction: We aimed to compare serum levels of Vitamin D3 in patients with and without Helicobacter pylori (HP) infec- tion detected with the upper gastrointestinal endoscopic examination. We hypothesized that patients diagnosed with HP pathologically had low serum vitamin D3 levels. Methods: The study was held between July 1, 2019, and March 31, 2021, tertiary referee hospital. The upper gastrointestinal system endoscopic data of pediatric patients with treatment-resistant dyspeptic complaints who were followed up in the Pediatric Gastroenterology outpatient clinic of hospital between July 1, 2019, and March 31, 2021, were retrospectively evaluated. The age, height, height standard deviation score (SDS), weight, weight SDS, body mass index (BMI), BMI SDS, and serum Vitamin D3 levels of the patients who were divided into two groups according to the presence of HP histopathologi- cally in the endoscopic biopsy samples were examined. Results: Eighty-six patients were evaluated, while 58 (67.44%) were girls and 28 (32.55%) were boys. The median age was 14.43 (ınterquartile range [IQR]=4.99) years. Histopathological evaluation of biopsy materials taken from the antrum and cor- pus of 43 patients who underwent upper gastrointestinal endoscopy revealed HP positivity in 43 patients (50.00%). HP was not detected in 43 patients (50.00%). The median Vitamin D3 level was 10.00 ng/ml in the HP positive group (IQR=6.00), and the median was 14.00 ng/ml (IQR=9.30) in the negative group. This study found a significant difference between HP positive and HP negative patient groups in Vitamin D3 serum levels (p=0.033). Discussion and Conclusion: Vitamin D3 serum levels were found to be significantly lower in the HP positive group than that of controls (p=0.033). This finding supports that mucosal damage may affect Vitamin D3 absorption. Early diagnosis and treatment maintain their importance to prevent long-term consequences of chronic infection.