Background Ependymin (Epd), the predominant protein in the cerebrospinal fluid of teleost fishes, was originally associated with neuroplasticity and regeneration. Ependymin-related proteins (Epdrs) have been identified in other vertebrates, including amphibians and mammals. Recently, we reported the identification and characterization of an Epdr in echinoderms, showing that there are ependymin family members in non-vertebrate deuterostomes. We have now explored multiple databases to find Epdrs in different metazoan species. Using these sequences we have performed genome mapping, molecular phylogenetic analyses using Maximum Likelihood and Bayesian methods, and statistical tests of tree topologies, to ascertain the phylogenetic relationship among ependymin proteins. Results Our results demonstrate that ependymin genes are also present in protostomes. In addition, as a result of the putative fish-specific genome duplication event and posterior divergence, the ependymin family can be divided into four groups according to their amino acid composition and branching pattern in the gene tree: 1) a brain-specific group of ependymin sequences that is unique to teleost fishes and encompasses the originally described ependymin; 2) a group expressed in non-brain tissue in fishes; 3) a group expressed in several tissues that appears to be deuterostome-specific, and 4) a group found in invertebrate deuterostomes and protostomes, with a broad pattern of expression and that probably represents the evolutionary origin of the ependymins. Using codon-substitution models to statistically assess the selective pressures acting over the ependymin protein family, we found evidence of episodic positive Darwinian selection and relaxed selective constraints in each one of the postduplication branches of the gene tree. However, purifying selection (with among-site variability) appears to be the main influence on the evolution of each subgroup within the family. Functional divergence among the ependymin paralog groups is well supported and several amino acid positions are predicted to be critical for this divergence. Conclusion Ependymin proteins are present in vertebrates, invertebrate deuterostomes, and protostomes. Overall, our analyses suggest that the ependymin protein family is a suitable target to experimentally test subfunctionalization in gene copies that originated after gene or genome duplication events.