Agarases are generally classified into glycoside hydrolase families 16, 50, and 86 and are found to degrade agarose to frequently generate neoagarobiose, neoagarotetraose, or neoagarohexaose as the main products. In this study we have cloned a novel endo-type beta-agarase gene, agaB, from marine Pseudoalteromonas sp. CY24. The novel agarase encoded by agaB gene has no significant sequence similarity with any known proteins including all glycoside hydrolases. It degrades agarose to generate neoagarooctaose and neoagarodecaose as the main end products. Based on the analyses of enzymatic kinetics and degradation patterns of different oligosaccharides, the agarase AgaB appears to have a large substrate binding cleft that accommodates 12 sugar units, with 8 sugar units toward the reducing end spanning subsites +1 to +8 and 4 sugar units toward the non-reducing end spanning subsites -4 to -1, and enzymatic cleavage taking place between subsites -1 and +1. In addition, 1H NMR analysis shows that this enzyme hydrolyzes the glycosidic bond with inversion of anomeric configuration, in contrast to other known agarases that are retaining. Altogether, AgaB is structurally and functionally different from other known agarases and appears to represent a new family of glycoside hydrolase.