The purpose of this study was to determine whether or not Helicobacter pylori (H. pylori) infection affects the hypothalamic-pituitary-adrenal (HPA) axis and to evaluate the association between vitamin D levels with H. pylori infected and eradicated patients. The glucagon stimulation test (GST) was used to assess the HPA gland axis. An H. pylori infection was diagnosed based on the rapid urease test and histology. All infected patients received triple eradication therapy. Three months after the treatment, 14C urea breath test was reinstituted, and GST was repeated in patients who were negative for H. pylori. Of the 43 patients, 20 (46%) were found to have a lower mean value of peak cortisol than normal responses to the GST in the H. pylori-infected subjects. In 29 subjects, H. pylori infection was successfully eradicated. Twelve (41%) out of 29 subjects were defined as having a blunted GST response and 17 (%59) subjects had normal HPA axis response to GST in the H. pylori-eradicated subjects. A significant correlation between low 25-hydroxyvitamin D3 (25(OH)D3) level and low peak cortisol response to GST was identified in the H. pylori-infected subjects. After H. pylori eradication, the positive correlation between 25(OH)D3 level and high peak cortisol response to GST was also identified. Mean 25(OH)D3 levels were lower in the H. pylori-infected subjects than in the H. pylori-eradicated subjects. Our results indicate an increased prevalence of blunted glucocorticoid response to GST in patients with infected H. pylori. Vitamin D deficiency is also common in the H. pylori-infected subjects and associated with blunted glucocorticoid response. These findings suggest that H. pylori eradication increases the cortisol response to GST. Vitamin D supports adrenal/cortisol production whereby a deficiency can result blunted glucocorticoid response to GST in patients with infected H. pylori.