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Small intestinal bacterial overgrowth (SIBO) occurs due to alteration of the microbiota within the upper gastrointestinal tract. Proton pump inhibitor (PPI) therapy has been suggested as a risk factor for SIBO; however, the published reports have yielded conflicting results on the association between PPI therapy and risk of developing SIBO. The aim of this study was to compare the prevalence of SIBO as measured by glucose hydrogen breath testing (GHBT) in patients on PPI therapy compared with those not on PPI therapy.A retrospective chart review was completed for all patients who underwent GHBT testing from 2004 to 2010. Breath samples for hydrogen (H2) and methane (CH4) were collected before and every 20 min for 120 min following ingestion of a 50-g oral glucose load. We used the following criteria to define a positive GHBT (a) increase in H2>20 parts per million (p.p.m.) over baseline, (b) sustained rise H2>10 p.p.m. over baseline, (c) CH4>15 p.p.m. over baseline, and (d) either rise H2>20 p.p.m. over baseline or CH4>15 p.p.m.A total of 1,191 patients (70% female) were included, of whom 566 (48%) were on PPI therapy. GHBT positivity did not differ significantly between PPI users and nonusers by any of the diagnostic criteria used and PPI use was not significantly associated with GHBT positivity using any of these criteria. GHBT positivity was associated with older age (odds ratio (OR) 1.03, 95% confidence interval (CI) 1.01–1.04) and antidiarrheal use (OR 1.99, 95% CI 1.15–3.44) using H2>20, older age (OR 1.01, 95% CI 1.00–1.02) and diarrhea (OR 1.53, 95% CI 1.13–2.09) using H2>10, and older age (OR 1.01, 95% CI 1.00–1.02) using either H2>20 or CH4>15. PPI use was not significantly associated with GHBT positivity using any of these criteria.In this large, adequately powered equivalence study, PPI use was not found to be significantly associated with the presence of SIBO as determined by the GHBT.