Thioredoxin binding protein‐2 (TBP‐2) is a negative regulator of thioredoxin and has multiple regulatory functions in cellular redox, growth, differentiation, apoptosis, and aging. To investigate the function of TBP‐2 in vivo, we generated mice with targeted inactivation of TBP‐2 (TBP‐2^−/− mice). Here, we show that TBP‐2 expression is markedly up‐regulated during fasting in wild‐type mice, while TBP‐2^−/− mice were predisposed to death with bleeding tendency, as well as hepatic and renal dysfunction as a result of 48 h of fasting. The fasting‐induced death was rescued by supplementation of glucose but not by that of oleic acid, suggesting that inability of fatty acid utilization plays an important role in the anomaly of TBP‐2^−/− mice. In these mice, plasma free fatty acids levels are higher, whereas glucose levels are lower than those of wild‐type mice. Compared with wild‐type mice, TBP‐2^−/− mice showed increased levels of plasma ketone bodies, pyruvate and lactate, indicating that Krebs cycle‐mediated fatty acid utilization is impaired. Because the fatal impairment of fatty acid utilization is a characteristically metabolic feature of Reye (‐like) syndrome, TBP‐2^−/− mouse may represent a novel model for investigating the pathophysiology of these disorders.