Introduction: The etiology of small intestinal bacterial overgrowth (SIBO) is diverse and frequently multi-factorial. SIBO is thought to result from a disruption of the normal peristalsis of the stomach and small intestine. It is currently unclear if hypochlorhydria secondary to proton pump inhibitors (PPIs) increases risk of SIBO; recent studies have yielded conflicting results regarding this association. To date, no published study has prospectively collected data regarding PPI dose and glucose hydrogen breath test results (GHBT). The goal of this study was to prospectively evaluate the association between small intestinal bacterial overgrowth (SIBO) and PPI use and dose using a GHBT. Methods: A questionnaire pertaining to PPI use was given to 65 consecutive patients prior undergoing GHBT. Breath samples for hydrogen (H2) and methane (CH4) were collected before and every 15 minutes for 180 minutes following ingestion of a 75-g oral glucose load. We used the following criteria to define a positive GHBT: (a) baseline H2 or CH4 over 10 parts per million (ppm), (b) rise in H2 or CH4 of at least 12 ppm after ingestion of the glucose load. All data were analyzed using SAS version 9.2 and R version 3.0.3. Results: The study included 65 patients (74% female; mean age 51 years), 24 of whom had a positive GHBT (37%). Of the patients with positive GHBT, 15 (62.5%) had an elevated baseline H2, 10 (41.6%) had an elevated baseline CH4, 8 (33.3%) had >12 ppm increase in H2 after oral glucose load, and 1 (4.2%) had >12 ppm increase in CH4 after oral glucose load. Current PPI use was not associated with positive GHBT when compared to patients not on PPI (45.4% vs. 32.6%, p=0.31). In the subgroup of patients who were exposed PPIs in the past 2 years, patients with positive GHBT were more likely to use it more than once daily (1.8 ± 0.71 vs. 1.1 ± 0.35; p=0.01). Conclusion: Current PPI use does not significantly increase risk of SIBO; however, use of PPI greater than once daily does increase the risk of SIBO. Further studies are needed to confirm the association between SIBO and PPI dosing.