ABSTRACT: Enhancers are distal cis-regulatory elements that activate the transcription of their target genes. They regulate a wide range of important biological functions and processes, including embryogenesis, development, and homeostasis. As more and more large-scale technologies were developed for enhancer identification, a comprehensive database is highly desirable for enhancer annotation based on various genome-wide profiling datasets across different species. Here, we present an updated database EnhancerAtlas 2.0 (http://www.enhanceratlas.org/indexv2.php), covering 586 tissue/cell types that include a large number of normal tissues, cancer cell lines, and cells at different development stages across nine species. Overall, the database contains 13 494 603 enhancers, which were obtained from 16 055 datasets using 12 high-throughput experiment methods (e.g. H3K4me1/H3K27ac, DNase-seq/ATAC-seq, P300, POLR2A, CAGE, ChIA-PET, GRO-seq, STARR-seq and MPRA). The updated version is a huge expansion of the first version, which only contains the enhancers in human cells. In addition, we predicted enhancer-target gene relationships in human, mouse and fly. Finally, the users can search enhancers and enhancer-target gene relationships through five user-friendly, interactive modules. We believe the new annotation of enhancers in EnhancerAtlas 2.0 will facilitate users to perform useful functional analysis of enhancers in various genomes.