Although many beta-agarases that hydrolyze the beta-1,4 linkages of agarose have been biochemically characterized, only three alpha-agarases that hydrolyze the alpha-1,3 linkages are reported to date. In this study, a new alpha-agarase, AgaWS5, from Catenovulum sediminis WS1-A, a new agar-degrading marine bacterium, was biochemically characterized. AgaWS5 belongs to the glycoside hydrolase (GH) 96 family. AgaWS5 consists of 1295 amino acids (140 kDa) and has the 65% identity to an alpha-agarase, AgaA33, obtained from an agar-degrading bacterium Thalassomonas agarivorans JAMB-A33. AgaWS5 showed the maximum activity at a pH and temperature of 8 and 40 degrees C, respectively. AgaWS5 showed a cold-tolerance, and it retained more than 40% of its maximum enzymatic activity at 10 degrees C. AgaWS5 is predicted to have several calcium-binding sites. Thus, its activity was slightly enhanced in the presence of Ca2+, and was strongly inhibited by EDTA. The K-m and V-max of AgaWS5 for agarose were 10.6 mg/mL and 714.3 U/mg, respectively. Agarose-liquefication, thin layer chromatography, and mass and NMR spectroscopic analyses demonstrated that AgaWS5 is an endo-type alpha-agarase that degrades agarose and mainly produces agarotetraose. Thus, in this study, a novel cold-adapted GH96 agarotetraose-producing alpha-agarase was identified.