Background: Omeprazole administration is associated with changes in gastric and fecal microbiota and increased incidence of Clostridioides difficile enterocolitis in humans and dogs. Hypothesis/Objectives: Study purpose was to assess the effect of omeprazole on gastric glandular and fecal microbiota in healthy adult horses. Animals: Eight healthy horses stabled on straw and fed 100% haylage. Methods: Prospective controlled study. Transendoscopic gastric glandular biopsies, gastric fluid, and fecal samples were obtained from each horse twice at a 7-day interval before the administration of omeprazole. Samples were taken on the same horses before and after a 7-day administration of omeprazole (4 mg/kg PO q24h). pH was assessed on fresh gastric fluid and other samples were kept at −20 C until analysis. Bacterial taxonomy profiling was obtained by V1V3 16S amplicon sequencing from feces and gastric glandular biopsies. Analysis of alpha, beta diversity, and comparison between time points were performed with MOTHUR and results were considered significant when P < .05. Results: Gastric pH increased significantly after 7 days of omeprazole administration (P = .006). Omeprazole did not induce significant major changes in composition of fecal or gastric glandular microbiota, however, after administration, certain microbial genera became more predominant in the gastric glandular mucosa (lower Simpson's evenness, P = .05). Only the genus Clostridium sensu strictu_1 had a significant shift in the glandular gastric mucosa after omeprazole administration (P = .002). No population shifts were observed in feces. Conclusions and Clinical Importance: Oral administration of omeprazole could have fewer effects in gastrointestinal microbiota in the horse compared to other species.