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We present a major update of MirGeneDB (3.0), the manually curated animal microRNA gene database. Beyond moving to a new server and the creation of a computational mirror, we have expanded the database with the addition of 33 invertebrate species, including representatives of 5 previously unsampled phyla, and 6 mammal species. MirGeneDB now contains entries for 21 822 microRNA genes (5160 of these from the new species) belonging to 1743 microRNA families. The inclusion of these new species allowed us to refine both the evolutionary node of appearance of a number of microRNA genes/families, as well as MirGeneDB's phylogenetically informed nomenclature system. Updated covariance models of all microRNA families, along with all smallRNA read data are now downloadable. These enhanced annotations will allow researchers to analyze microRNA properties such as secondary structure and features of their biogenesis within a robust phylogenetic context and without the database plagued with numerous false positives and false negatives. In light of these improvements, MirGeneDB 3.0 will assume the responsibility for naming conserved novel metazoan microRNAs. MirGeneDB is part of RNAcentral and Elixir Norway and is publicly and freely available at mirgenedb.org., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)