Understanding the selectivity of aquaporin (AQP) membrane channels will require structural and functional studies of wild type and modified proteins; however, expression systems have not previously yielded AQPs in the necessary milligrams quantities. Cell free (CF) systems have emerged in recent years as fast, efficient and versatile technologies for the production of membrane proteins. Here, we establish a convenient method to make large amounts of efficient human AQP3, a physiologically relevant aquaglyceroporin permeating glycerol, water and urea.AQP3 was produced in an E. coli extract based CF system using the D-CF mode (CF membrane protein expression in presence of detergent). Milligrams amounts of a recombinant histidine-tagged AQP3 protein (hAQP3-6His) per ml of CF reaction were synthesized in presence of the nonionic detergent Brij 98 and purified by Ni-NTA chromatography. Purified hAQP3-6His was incorporated into liposomes and analyzed functionally by stopped flow light scattering. The glycerol permeability (Pgly) of the proteoliposomes resulted four times higher than the Pgly of the empty (control) liposomes (7.08±0.7 vs 1.74±0.3 x 10-3 mm/s, respectively). Consistently, the Pgly of the proteoliposomes was markedly reduced (-50%) after incubation with 0.5 mM phloretin, a known AQP3 blocker. AQP3 is the first aquaglyceroporin produced in preparative scale by a CF system, which could serve as basis for further structural/functional studies and biomimetic technologies.