The enzymatic hydrolysis of the glycosidic bond is central to numerous biological processes. Glycoside hydrolases, which catalyze these reactions, are grouped into families based on primary sequence similarities. One of the largest glycoside hydrolase families is glycoside hydrolase family 5 (GH5), which contains primarily endo-acting enzymes that hydrolyze ␤-mannans and ␤-glucans. Here we report the cloning, characterization, and three-dimensional structure of the Cellvibrio mixtus GH5 ␤-mannosidase (CmMan5A). This enzyme releases mannose from the nonreducing end of mannooligosaccharides and polysaccharides, an activity not previously observed in this enzyme family. CmMan5A contains a single glycone (؊1) and two aglycone (؉1 and ؉2) sugarbinding subsites. The ؊1 subsite displays absolute specificity for mannose, whereas the ؉1 subsite does not accommodate galactosyl side chains but will bind weakly to glucose. The ؉2 subsite is able to bind to decorated mannose residues. CmMan5A displays similar activity against crystalline and amorphous mannans, a property rarely attributed to glycoside hydrolases. The 1.5 Å crystal structure reveals that CmMan5A adopts a (␤/␣) 8 barrel fold, and superimposition with GH5 endomannanases shows that dramatic differences in the length of three loops modify the active center accessibility and thus modulate the specificity from endo to exo. The most striking and significant difference is the extended loop between strand ␤8 and helix ␣8 comprising residues 378-412. This insertion forms a "double" steric barrier, formed by two short ␤-strands that function to "block" the substrate binding cleft at the edge of the ؊1 subsite forming the "exo" active center topology of CmMan5A.