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Proton pump inhibitors (PPI), used to treat gastrointestinal disorders, are associated with alterations in the gut microbiome. However, this is understudied in Puerto Ricans who have unique lifestyle characteristics. Puerto Ricans, including participants of the Boston-Puerto Rican Health Study (BPRHS), report high PPI use. Therefore, we examined gut microbial variations associated with PPI use in the BPRHS. BPRHS is a prospective cohort. 309 BPRHS participants self-reported PPI use and self-collected, metagenomically profiled, stool samples. PPI use was classified as any use in the past 30 days. Cross-sectional associations between gut microbial taxa, functional pathways, and PPI use were examined using omnibus analyses, multivariate linear modeling in MaAsLin2, and random forest classifier in feature-wise analyses. We further compared our results with the non-Hispanic Health Professionals Follow-Up Study (HPFS) to validate key findings and examine ethnicity-related differences. Among 309 participants (mean age 68.8 years; female 74.6%), 112 (36%) self-reported PPI use. After adjusting for relevant covariates, we observed an enrichment of Streptococcus parasanguinis (β = 3.16, FDR p = 0.01), S. anginosus (β = 2.89, FDR p < 0.01), S. salivarius (β = 2.56, FDR p = 0.01), S. gordonii (β = 1.98, FDR p = 0.15), and Rothia mucilaginosa (β = 1.54, FDR p = 0.06), among PPI users compared to non-users. Streptococci, Lactobacilli, and Enterococci predominantly contributed to the functional pathways associated with PPI use. The observed enrichment of oral-typical taxa, such as Streptococci, among PPI users in the BPRHS suggests the potential of PPIs to alter gut microbial composition. More studies are needed to understand the impact of PPI use on the gut microbiome in different ethnicities. Trial Registration: Parent study (BPRHS) NCT01231958.