Objective: Nitrofurantoin is a synthetic antibiotic and is used for treatment and prevention of urinary tract infection. The common side effect of nitrofurantoin is lung disorders, and some studies reported its mitochondrial toxicity. The aim of this study was to investigate the protective effect of dithioteritol (DTT) as a thiol reductant against mitochondrial toxicity induced by nitrofurantoin. Materials and Methods: Mitochondrial viability was assessed by 3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyl tetrazolium bromide (MTT) assay. Also in this study the effect of nitrofurantoin on superoxide dismutase (SOD) activity, ATP production, mitochondrial swelling and oxidative stress was investigated. Results: Our results showed that nitrofurantoin (0.1-10 µM) reduced viability of mitochondria in a dose- and time-dependent manner. Nitrofurantoin induced lipid peroxidation and oxidative stress in mitochondria and depleted mitochondrial ATP. DTT (0.5 mM) reduced toxicity of nitrofurantoin. Conclusion: Toxicity of nitrofurantoin is mediated by one electron reduction in redox cycle and induction of oxidative stress. DTT can also reduce thiol and non-thiol proteins and prevent nitrofurantoin toxicity.