First introduced in 2021, MetOrigin has quickly established itself as a powerful web server to distinguish microbial metabolites and identify the bacteria responsible for specific metabolic processes. Building on the growing understanding of the interplay between the microbiome and metabolome, and in response to user feedback, MetOrigin has undergone a significant upgrade to version 2.0. This enhanced version incorporates three new modules: (1) Quick search module that facilitates the rapid identification of bacteria associated with a particular metabolite; (2) Orthology analysis module that links metabolic enzyme genes with their corresponding bacteria; (3) Mediation analysis module that investigates potential causal relationships among bacteria, metabolites, and phenotypes, highlighting the mediating role of metabolites. Additionally, the backend MetOrigin database has been updated with the latest data from seven public databases (KEGG, HMDB, BIGG, ChEBI, FoodDB, Drugbank, and T3DB), with expanded coverage of 210,732 metabolites, each linked to its source organism. MetOrigin 2.0 is freely accessible at http://metorigin.met-bioinformatics.cn.