By searching the PubMed database, we manually curated lists of R-loop regulators for multiple species. As of March 31, 2021, we had collected 1,185 regulators in human, 24 in mouse, 63 in yeast (Saccharomyces cerevisiae) and 21 in E. coli. The curation has now been updated through December 31, 2025, resulting in a current collection of 1,709 human, 76 mouse, 77 yeast and 23 E. coli R-loop regulatory proteins. In addition, we revised the functional annotations and related information for 610 human, 6 mouse, 8 yeast and 3 E. coli regulators that were previously cataloged. Examplified with validated human R-loop regulators, we performed PPI network analysis to provide a global overview of functional relationship of R-loop regulators. Seven distinct clusters were readily identified, and GO enrichment analysis revealed cluster-specific enrichment of biological processes.