BACKGROUND: In this study, we aimed to characterize the gut microbiome and its potential functioning in 2 populations at 2 time points during pregnancy in relation to mental distress. METHODS: During the second and third trimester, individuals from the United States and Sweden completed the Edinburgh Postnatal Depression Scale and provided fecal samples for whole-genome metagenomics. A total of 832 and 161 samples were sequenced and analyzed from the Swedish cohort and the U.S. cohort, respectively. Multiple characterizations of the microbial community were analyzed in relation to distress measured using the Edinburgh Postnatal Depression Scale. Principal coordinate analysis and distance-based redundancy analysis assessed variation in functional gut-brain modules. For the U.S. cohort, the Trier Social Stress Test was administered 8 weeks postpartum while collecting salivary cortisol. RESULTS: Principal coordinate analysis identified 4 sample clusters based on the gut-brain modules distinguished by functions such as short-chain fatty acid synthesis and cortisol degradation. While with distance-based redundancy analysis, mental distress subtypes did not significantly contribute to variation in gut-brain modules (p = .085 for Sweden, p = .23 for the U.S.), a U.S. sample cluster distinguished by lower cortisol degradation from another cluster with higher gut microbial cortisol degradation abundance had significantly higher odds of being associated with depression (p = .024). The U.S. sample cluster with lower gut microbial cortisol degradation abundance also had significantly higher cortisol levels after a postpartum social stressor. CONCLUSIONS: Further studies are warranted to investigate the potential for the gut microbiome to serve as biomarkers of gut-brain axis health during pregnancy across disparate populations.