The adult form of Sandhoff disease with the motor neuron disease phenotype is a rare neurodegenerative disorder caused by mutations in HEXB encoding the β-subunit of β-hexosaminidase, yet the properties of mutant β-subunits of the disease have not been fully determined. We identified a novel mutation (H235Y) in the β-sheet of the (β/α)₈-barrel domain, in addition to the previously reported P417L mutation that causes aberrant splicing, in a Japanese patient with the motor neuron disease phenotype. Enzyme assays, gel filtration studies and immunoprecipitation studies with HEK293 cells transiently expressing mutant β-subunits demonstrated that the H235Y mutation abolished both α-β and β-β dimer formation without increasing β-hexosaminidase activity, whereas other reported mutant β-subunits (Y456S, P504S or R533H) associated with the motor neuron disease phenotype formed dimers. Structural analysis suggested that the H235Y mutation in the β-sheet of the (β/α)₈-barrel domain changed the conformation of the β-subunit by causing a clash with the E288 side chain. In summary, H235Y is the first mutation in the β-sheet of the (β/α)₈-barrel domain of the β-subunit that abolishes α-β and β-β dimer formation; the presented patient is the second patient to exhibit the motor neuron disease phenotype with P417L and a non-functional allele of HEXB.