Abstract Background Extremophiles evolved capacities to survive extended exposure to harsh environmental conditions such as complete desiccation (anhydrobiosis) and freezing (cryobiosis). Accumulation of the three-carbon polyhydric alcohol glycerol is commonly observed in anhydrobiotic organisms, although it is considered to preferentially enhance cryobiosis rather than anhydrobiosis. Results Here, using dormant stages of the halophilic extremophile crustacean Artemia franciscana, we show that this role is reversed. We find that A. franciscana and related branchiopods evolved co-opted entomoglyceroporin (Eglp)-like aquaporin-type channels previously only characterized in hexapods. Phylogenomic and site-directed mutagenesis analyses indicate that EglpL orthologs likely evolved during the early Cambrian in the common ancestor of the Pancrustacea. RNAi-mediated knockdown experiments show that the A. franciscana EglpL glycerol transporter is subfunctionally co-regulated with canonical aquaglyceroporins (Glps) to mediate glycerol accumulation in the diapause cysts. Termination of diapause using either desiccation or hydrogen peroxide and further exposure of the cysts to freezing suggest that the acquired glycerol plays a more critical role in anhydrobiosis rather than cryobiosis. Conclusions These findings uncover the essential role of evolutionary divergent aquaporin-type glycerol channels in the accrual of glycerol in an anhydrobiotic organism and reveal a previously overlooked function of this polyol for desiccation tolerance.