miRWalk2.0 is a comprehensive database containing the largest collection of predicted and experimentally verified microRNA (miRNA)-target interactions, with approximately 949 million entries. It improves upon the previous version by incorporating 13 prediction data sets from existing miRNA-target resources. The web interface provides a multilayered view of data, including information on miRNAs, genes, epigenomics, pathways, ontologies, protein classes, phenotype, genotype, single-nucleotide polymorphisms, functional networks, tandem mass spectra, and relevant PubMed articles. Users can also download information on human homologous genes across 15 species for interspecies analysis. Additionally, users can create lists of genes or their identifiers for external tools like DAVID. miRWalk2.0 offers a framework for obtaining statistically significant miRNA interactions associated with pathways, ontologies, gene and protein classes, disorders, and mitochondrial genomes. It also includes experimentally verified information on miRNAs linked with genes, diseases, pathways, ontologies, cell lines, and organs. A novel feature called "Customized data set" allows users to generate a customized list of putative targets from 13 different data sets. miRWalk2.0 provides a platform for a comparative overview of miRNA-lncRNA interactions. Gene- and lncRNA-based searches help identify targets and nontargets for queried miRNAs, reducing false positives. It also allows genomic location searches to determine miRNAs sharing the same or nearby locations. This information can be used to validate other miRNAs involved in genetic regulation. The validated-target module is updated every 3 months, while the predicted-target module is updated twice a year. The current status of miRWalk2.0 is detailed in Table 1. More annotations and additional species will be integrated to expand this resource. The authors declare no competing financial interests.miRWalk2.0 is a comprehensive database containing the largest collection of predicted and experimentally verified microRNA (miRNA)-target interactions, with approximately 949 million entries. It improves upon the previous version by incorporating 13 prediction data sets from existing miRNA-target resources. The web interface provides a multilayered view of data, including information on miRNAs, genes, epigenomics, pathways, ontologies, protein classes, phenotype, genotype, single-nucleotide polymorphisms, functional networks, tandem mass spectra, and relevant PubMed articles. Users can also download information on human homologous genes across 15 species for interspecies analysis. Additionally, users can create lists of genes or their identifiers for external tools like DAVID. miRWalk2.0 offers a framework for obtaining statistically significant miRNA interactions associated with pathways, ontologies, gene and protein classes, disorders, and mitochondrial genomes. It also includes experimentally verified information on miRNAs linked with genes, diseases, pathways, ontologies, cell lines, and organs. A novel feature called "Customized data set" allows users to generate a customized list of putative targets from 13 different data sets. miRWalk2.0 provides a platform for a comparative overview of miRNA-lncRNA interactions. Gene- and lncRNA-based searches help identify targets and nontargets for queried miRNAs, reducing false positives. It also allows genomic location searches to determine miRNAs sharing the same or nearby locations. This information can be used to validate other miRNAs involved in genetic regulation. The validated-target module is updated every 3 months, while the predicted-target module is updated twice a year. The current status of miRWalk2.0 is detailed in Table 1. More annotations and additional species will be integrated to expand this resource. The authors declare no competing financial interests.