Laparoscopic Roux-en-Y gastric bypass (LRYGB) achieves sustainable weight loss possibly by altering the gut microbiota. The effect of a proton pump inhibitor (PPI) on weight loss and the gut microbiota has not been explored. PPI use and the gut microbiota were assessed before and 6 months after LRYGB in eight patients. Bacterial profiles were generated by 16S ribosomal RNA (rRNA) gene sequencing. Prior to LRYGB, PPI users had a higher percent relative abundance (PRA) of Firmicutes compared to nonusers. PPI users at 6 months post-LRYGB had a higher PRA of Firmicutes [48.6 versus 35.6%, p = nonsignificant (NS)] and a trend toward significantly lower percent excess weight loss (49.3 versus 61.4%, p = 0.067) compared to nonusers. PPI use post-LRYGB may impair weight loss by modifying gut microbiota.