In our screening of plant growth regulators of microbial origin, rotihibin B (1), a minor component, was isolated from the culture filtrate of Streptomyces sp. strain 3C02 along with the active substance rotihibin A (2) (structure preliminarily reported). We elucidated the structure of 1 by tandem mass spectrometry. High-resolution FABMS indicated the molecular formula of 1 as C35H62N10O14 (MW 846), compared to C35H63N11O13 (MW 845) for 2, suggesting an NH group in 2 was replaced by O in 1. Tandem mass spectra showed N-terminal ions (B5, C5) and C-terminal ions (Y2, Y3, Y4) of 1 shifted by one mass unit from those of 2, indicating the β-hydroxyasparagine residue in 2 was replaced by β-hydroxyaspartic acid in 1. Thus, the structure of 1 was deduced. Tandem mass spectrometry proved useful for sequence analysis of such peptides.