Bletilla striata is consumed as food and herbal medicine. Militarine (MLT) is a major ingredient in B. striata. Previous studies demonstrated that MLT showed teratogenic toxicity to zebrafish embryos. The present study aimed to identify reactive metabolites possibly involved in the cytotoxicity of MLT and determine the metabolic pathways involved. MLT was found to be hydrolyzed to p-hydroxybenzyl alcohol (HBA) by β-glucosidase and esterases. The resulting HBA further underwent spontaneous dehydration to form quinone methide. HBA was also metabolized to the corresponding sulfate, followed by departure of the sulfate to generate a quinone methide. The resultant quinone methide reacted with hepatic glutathione (GSH) and protein to form the corresponding GSH conjugate and protein adduction. Additionally, inhibition of sulfotransferases (SULTs) attenuated the susceptibility of hepatocytes to the toxicity of MLT. This study provides that the hydrolytic enzymes β-glucosidase, esterases, and SULTs participate in the metabolic activation of MLT.