Aquaporins are integral membrane proteins that facilitate the diffusion of water and other small, uncharged solutes across the cellular membrane and are widely distributed in organisms from humans to bacteria. However, the characteristics of prokaryotic aquaporins remain largely unknown. We investigated the distribution and sequence characterization of aquaporins in prokaryotic organisms and summarized the transport characteristics, physiological functions, and regulatory mechanisms of prokaryotic aquaporins. Aquaporin homologues were identified in 3315 prokaryotic genomes retrieved from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, but the protein clustering pattern is not completely congruent with the phylogeny of the species that carry them. Moreover, prokaryotic aquaporins display diversified aromatic/arginine constriction region (ar/R) amino acid compositions, implying multiple functions. The typical water and glycerol transport characterization, physiological functions, and regulations have been extensively studied in Escherichia coli AqpZ and GlpF. A Streptococcus aquaporin has recently been verified to facilitate the efflux of endogenous H2O2, which not only contributes to detoxification but also to species competitiveness, improving our understanding of prokaryotic aquaporins. Furthermore, recent studies revealed novel regulatory mechanisms of prokaryotic aquaporins at post-translational level. Thus, we propose that intensive investigation on prokaryotic aquaporins would extend the functional categories and working mechanisms of these ubiquitous, intrinsic membrane proteins.