Objective To identify and characterize a new beta-agarase fromCellulophaga omnivescoriaW5C capable of producing biologically-active neoagarooligosaccharides from agar. Results The beta-agarase, Aga1, has signal peptides on both N- and C-terminals, which are involved in the type IX secretion system. It shares 75% protein sequence identity with AgaD fromZobellia galactanivoransand has a molecular weight of 54 kDa. Biochemical characterization reveals optimum agarolytic activities at pH 7-8 and temperature 30-45 degrees C. Aga1 retains at least 33% activity at temperatures lower than the sol-gel transition state of agarose. Metal ions are generally not essential, but calcium and potassium enhance its activity whereas iron and zinc are inhibitory. Finally, hydrolysis of agarose with Aga1 yields neoagarotetraose, neoagarohexaose, and neoagarooctaose. Conclusions Aga1 displays unique traits such as moderate psychrophilicity, stability, and synergy with other agarases, which makes it an excellent candidate for biosynthetic production of neoagarooligosaccharides from agar.