The　relationship　between　unfolding　and　inactivation　of　Hypocrea　orientalis　β-glucosidase　has　been　investigated　for　the　first　time.　The　secretion　of　β-glucosidase　from　H.　orientalis　is　induced　by　raw　cassava　residues.　The　enzyme　was　75　kD　without　glycosylation.　Guanidine　hydrochloride　(GuHCl)　could　reversibly　inactivate　the　enzyme　with　an　estimated　IC50　value　of　0.4　M.　The　inactivation　kinetics　model　by　GuHCl　has　been　established　and　the　microscopic　inactivation　rate　constants　are　determined.　The　values　of　forward　inactivation　rate　constants　of　free　enzyme　are　found　to　be　larger　than　that　of　substrate–enzyme　complex　suggesting　the　enzyme　could　be　protected　by　substrate　during　denaturation.　Conformational　change　of　the　enzyme　during　denaturation　is　observed　as　the　intrinsic　fluorescence　emission　peaks　appeared　red-shift　(334–354　nm)　with　intensity　decreased　following　increase　of　GuHCl　concentrations.　Inactivation　extent　is　found　to　be　greater　than　conformation　change　of　the　whole　enzyme,　indicating　that　the　active　site　of　H.　orientalis　β-glucosidase　might　be　a　more　flexible　region　than　the　whole　enzyme.　©　2017　The　Society　for　Biotechnology,　Japan