There is currently a major gap in understanding the physiological processes of tissue grade animals from early diverging phyla, particularly within the context of the molecular mechanisms in response to osmotic variation. A key protein family essential for the regulation of intracellular water and solute concentrations are the Major Intrinsic Proteins (MIPs), these include aquaporins (Aqps) and aquaglyceroporins (Glps). Here, we report the identification and functional analysis of MIPs in Nematostella vectensis, a member of the phylum Cnidaria that represents a unique group of tissue grade organisms. Using a combination of sequence similarity and phylogenetic analyses, we successfully identified eight MIPs in N.vectensis, 4 Aqps, 3 Glps, and 1 in the atypical MIP groups 11/12. Putative MIPs were recombinantly expressed in budding yeast, Saccharomyces cerevisiae, and tested for functionality under osmotic stress. N. vectensis MIPs displayed varying degrees of functionality, ranging from no effect indicating lack in functionality to lethality indicating MIP functional capacity to translocate water, suggesting a phylogenetic approach paired with an in vivo functionality assay is critical for their characterization. Total N. vectensis MIP transcripts were also quantified during acute and long-term salinity exposure that support the results from the yeast experiments. Furthermore, based on high confidence protein structure homology modeling, we demonstrate a significant correlation between three biophysical pore properties that accurately predict functionality. The identification and functional analysis of these MIPs suggest a high degree of diversification at the cnidarian-bilaterian ancestor both in terms of number and function.