Many agarolytic bacteria degrade agar polysaccharide into the disaccharide unit neoagarobiose [O-3,6-anhydro-alpha-L-galactopyranosyl-(1-->3)-D-galactose] using various beta-agarases. Neoagarobiose hydrolase is an enzyme that acts on the alpha-1,3 linkage in neoagarobiose to yield D-galactose and 3,6-anhydro-L-galactose. This activity is essential in both the metabolism of agar by agarolytic bacteria and the production of fermentable sugars from agar biomass for bioenergy production. Neoagarobiose hydrolase from the marine bacterium Saccharophagus degradans 2-40 was overexpressed in Escherichia coli and crystallized in the monoclinic space group C2, with unit-cell parameters a = 129.83, b = 76.81, c = 90.11 A, beta = 101.86 degrees . The crystals diffracted to 1.98 A resolution and possibly contains two molecules in the asymmetric unit.