An agar-degrading Antarctic bacterium was isolated and identified as Pseudoalteromonas sp. NJ21. This strain showed strong agarolytic activity and could use agar as the sole carbon source for growth. A novel β-agarase gene aga3463 was cloned and identified from the genomic library of Pseudoalteromonas sp. NJ21. It was predicted to encode a peptide of 366 amino acids， including a 21-amino-acid signal peptide. BLAST analysis revealed the deduced amino acid sequence of aga3463 shared less than 36% similarity with any characterized agarase， and phylogenetic analysis showed it belonged to the glycoside hydrolase GH86 family. The recombinant Aga3463 protein had optimal temperature and pH of 50 °C and 7.0， respectively， and retained more than 60% activity from 10 to 50 °C. The metal ions Cd， Ca， Fe， and Mn increased Aga3463 activity， whereas Cu， Si， Fe， and Ni decreased Aga3463 activity. Notably， Aga3463 exhibited good thermostability in the presence of Ca; however， Ca was not necessary for its catalytic activity. The K and V values for Aga3463 were 6.17 mg/ml and 557 U/mg， respectively. Thin layer chromatography， liquid chromatography-mass spectrometry， nuclear magnetic resonance spectroscopy， and enzyme assay using p-nitrophenyl-α/β-d-galactopyranoside revealed Aga3463 as an exo-type β-agarase with the ability to degrade agarose to neoagarobiose as the major end product.