Most of the reported [13C]-urea breath test procedures use a test meal, which is believed to assist in the spread of the [13C]-urea solution into the entire stomach, as results without a test meal may mainly reflect urease activity in the antrum. Yet, procedures for the [13C]-urea breath test and interpretation of the obtained 13C excess value have not been well established. We carried out the present study to validate the usefulness of the [13C]-urea breath test in fasting subjects and to establish cut-off values. [13C]-urea breath tests were performed on 258 Helicobacter pylori-positive and 151 -negative subjects (247 H. pylori positive and 26 negative prior to any H. pylori cure treatment and 125 H. pylori negative and 11 positive after undergoing H. pylori cure treatment). The breath test procedure was performed under the following conditions: an 8 h fast, mouth washing before and after dosing, administration of 100 mg [13C]-urea, collection of breath sample in a plastic bag, a baseline and a 20 min sampling point and subject in a sitting position. Delta-13C at the 20 min sampling point in H. pylori-positive and -negative subjects was 31.0 ± 1.25 and 1.6 ± 0.11‰, respectively. Although the mean Δ13C value was greatest in duodenal ulcer or ulcer scar patients, there were no significant differences among mean Δ13C values in the various diseases. From Receiver Operator Characteristic curves and calculation of accuracy of the test, a cut-off value of 5.0‰ is considered to be appropriate for diagnosis of H. pylori infection, which provides 96.7% specificity and 96.5% sensitivity, suggesting that the [13C]-urea breath test in the fasting state is as effective in detecting the presence of H. pylori as other reported methods.