Nitrofurantoin (NFT) is a widely used antimicrobial agent in the treatment of specific urinary tract infections (UTIs). Many adverse effects associated with NFT use have been reported, including hepatotoxicity. A structure-toxicity relationship study was performed to gain the insight into the mechanisms of toxic action of NFT. The toxic effects of NFT and its nine analogues or constituent moieties (1-9) designed and synthesized by structural manipulation of NFT were evaluated in rat liver microsomes and primary rat hepatocytes. A decrease in ability to deplete glutathione (GSH) was found in the following order: nitrofuran-containing compounds (NFT and 1-3) > nitrobenzene-containing compounds (4 and 5) > nitro-free compounds (6-9). A similar pattern was observed in the cytotoxicity of these compounds as that of GSH depletion. The potential for reduction (electron deficiency) of nitro groups of the nitro-containing test compounds (NFT, 1-5) decreased with the decrease in the ability to deplete GSH and the intensity of their cytotoxicity. The corresponding nitroso and hydroxylamine intermediates resulting from metabolic reduction of NFT were found to be reactive to GSH for the first time. Additionally, nitro-containing compound 4 (a model compound) was much more cytotoxic than the corresponding analine (4a). The findings allowed us not only to define the mechanism of toxic action of NFT but also to provide medicinal chemists with instructive guidance for rational design of nitro-containing pharmaceutical agents.