Allosamidin, an inhibitor of Bombyx chitinase isolated from Streptomyces sp., consists of two N-acetylallosamine units and the oxazoline derivative allosamizoline (1). During the purification of 1, a minor active component was isolated and identified as demethylallosamidin (2), a demethyl derivative of 1. This study reports the structure and chitinase inhibitory activity of 2. The structure of 2 was determined using FAB mass spectrometry (FABMS) and nuclear magnetic resonance (NMR) spectroscopy: FABMS revealed a (M+H)+ ion at m/z 609, 14 mass units less than that of 1; 1H-NMR showed a single N-methyl signal at δ3.00 ppm (compared to two signals at δ3.17 ppm in 1); and 13C-NMR detected one N-methyl carbon at δ29.1 (versus two at δ38.4 in 1), confirming 2 as a demethyl derivative of 1. Demethylallosamidin (2) strongly inhibited Bombyx chitinase with an IC50 value of 0.25 μg/ml, which is comparable to the IC50 of 0.20 μg/ml for 1 (using a modified assay with Chitin Azure). Pseudodisaccharide 3, obtained by mild hydrolysis of 1, exhibited similar inhibitory activity to 1 and 2, while allosamizoline showed no activity. These findings indicate that the glycosidic bond between allosamizoline and N-acetylallosamine is essential for the biological activity, likely mimicking the glycosidic bond of chitin.