Fear is prone to return following extinction that is the basis of exposure therapy for fear-related disorders. Manipulations that enhance the extinction process can be beneficial for treatment. Animal studies have shown that fasting or caloric restriction can enhance extinction and inhibit the return of fear. The present study examined the effects of fasting on fear acquisition, extinction, and the return of fear in humans. One hundred and twenty-five male participants were randomized into a fasting group and food group and exposed to a Pavlovian fear conditioning paradigm. Changes in plasma cortisol and ghrelin levels were examined using enzyme-linked immunosorbent assays. One-night fasting had no effect on fear acquisition but enhanced fear extinction retention and prevented the return of fear, and this effect persisted for at least 6 months. This procedure was also effective for remote fear memory. Plasma ghrelin levels were elevated after fasting and had a negative relationship with the fear response in spontaneous recovery test. However, overnight fasting did not affect cortisol levels. These findings indicate that fasting enhances extinction retention and prevents the return of fear, without influencing fear memory formation. We propose that this novel procedure may open new avenues for promoting extinction-based therapies for fear-related disorders.