Bromoconduritol (6-bromo-3,4,5-trihydroxycyclohex-1-ene; BCD) has been known as an inhibitor of glucosidase II (G-II), which plays pivotal roles in glycoprotein processing and folding in the ER. Previous works suggested that BCD specifically inhibits the cleavage of the innermost glucose (Glc) among two alpha1-3 linked Glc residues (cleavage-2). This study addressed the mode of BCD's inhibition toward G-II by using fluorescently labeled substrates. Our analysis clarified that BCD inhibits both cleavage-1 and cleavage-2 activities of G-II. However, the inhibitory activity toward cleavage-2 was 6-fold higher than that toward cleavage-1. Inhibition against both of these activities was retained after dialysis, supporting that BCD exhibits inhibition through irreversible binding to G-II.