Background and Aims: Recent evidence has implicated the gut microbiome in the regulation of host metabolites and the pathogenesis of metabolic dysfunction–associated steatotic liver disease (MASLD). However, the role of gut microbiome and its associated metabolites as regulators and/or modulators in MASLD remains understudied. We aimed to investigate the gut microbiome diversity and composition, its interaction with plasma metabolites, and MASLD risk using population-level data. Methods: We combined 16S rRNA sequencing of stool samples with a wide range of plasma metabolites measured by the Metabolon HD4 platform, and MASLD diagnosed by abdominal ultrasound in the Rotterdam Study cohort at baseline, between 2012 and 2014. We assessed the relationship of gut microbiome and metabolites with MASLD separately. Then, the metabolic profile as exposure was obtained through multivariate linear or logistic regression models for MASLD, liver fibrosis, alpha diversity, and MASLD-associated genera. Results: Our gut microbiome analysis showed that alpha diversity was lower in MASLD individuals (Shannon: P value = 2.06 × 10−6), beta diversity differed across MASLD strata (R2 = 0.00158, P value <.001), and Lachnospiraceae UCG-010 (odds ratio = 1.14 (1.06–1.23), area under the curve = 0.7807)) was significantly associated with MASLD, after multiple testing correction (q value<0.05). The metabolomics analysis showed 160 out of the 991 studied metabolites to be significantly associated with MASLD (q value<0.05). Moreover, 57 of the identified metabolites were significantly correlated with gut microbiome at the genus level in individuals with MASLD. Conclusion: This study indicates alterations in several plasma metabolites to be linked to gut microbiome in MASLD. These results lay the ground for future studies to better understand the host-gut microbiota metabolic interactions in the development of MASLD.