N-glycosylation has been shown to be important for the stability of some glycoproteins. Isopullulanase (IPU), a polysaccharide-hydrolyzing enzyme, is a highly N-glycosylated protein, and IPU deglycosylation results in a decrease in thermostability. To investigate the function of N-glycan in IPU, we focused on an N-glycosylated residue located in the vicinity of the active site, Asn448. The thermostabilities of three IPU variants, Y440A, N448A and S450A, were 0.5–8.4°C lower than the wild-type enzyme. The crystal structure of endoglycosidase H (Endo H)-treated N448A variant was determined. There are four IPU molecules, Mol-A, B, C and D, in the asymmetric unit. The conformation of a loop composed of amino acid residues 435–455 in Mol-C was identical to wild-type IPU, whereas the conformations of this loop in Mol-A, Mol-B and Mol-D were different from each other. These results suggest that the Asn448 side chain is primarily important for the stability of IPU. Our results indicate that mutation of only N-glycosylated Asn residue may lead to incorrect conclusion for the evaluation of the function of N-glycan. Usually, the structures of N-glycosylation sites form an extended configuration in IPU; however, the Asn448 site had an atypical structure that lacked this configuration.