The pseudo-oligosaccharides, validamycins, showed potent inhibitory activity against trehalase of Rhizoctonia solani while no significant inhibition was exhibited against cellulase, pectinase, chitinase, α-amylase, α- and β-glucosidases. In particular, validoxylamine A strongly inhibited trehalase in a competitive manner with a Ki value of 1.9×10^-9M. The uptake of the antibiotic into the cell and the amount of the intracellular trehalose were investigated by incubating the washed mycelia of R. solani with validamycins. It was found that validamycin A is transported into the cell and hydrolyzed therein by a β-glucosidase yielding validoxylamine A with greater inhibitory activity. Also validamycin A containing β-D-glucosyl residue is more favorably taken up into the cell than validamycin D containing α-D-glucosyl residue or their common aglycone, validoxylamine A. In addition, validamycin A suppressed the in vivo degradation of the intracellular trehalose at very low concentration of 0.1 μg/ml.