Fear memories are critical for survival. Nevertheless, over-generalization of these memories, depicted by a failure to distinguish threats from safe stimuli, is typical in stress-related disorders. Previous studies have supported a protective role of ketamine against stress-induced depressive behavior. However, the effect of ketamine on fear generalization remains unclear. In this study, we investigated the effects of ketamine on fear generalization in a fear-generalized mouse model. The mice were given a single sub-anesthetic dose of ketamine (30 mg/kg, i.p.) 1 h before, 1 week before, immediately after, or 22 h after fear conditioning. The behavioral measure of fear (indicated by freezing level) and synaptic protein expression in the basolateral amygdala (BLA) and inferior-limbic pre-frontal cortex (IL-PFC) of mice were examined. We found that only ketamine administered 22 h after fear conditioning significantly decreased the fear generalization, and the effect was dose-dependent and lasted for at least 2 weeks. The fear-generalized mice showed a lower level of brain-derived neurotrophic factor (BDNF) and a higher level of GluN2B protein in the BLA and IL-PFC, and this was reversed by a single administration of ketamine. Moreover, the GluN2B antagonist ifenprodil decreased the fear generalization when infused into the IL-PFC, but had no effect when infused into the BLA. Infusion of ANA-12 (an antagonist of the BDNF receptor TrkB) into the BLA or IL-PFC blocked the effect of ketamine on fear generalization. These findings support the conclusion that a single dose of ketamine administered 22 h after fear conditioning alleviates the fear memory generalization in mice and the GluN2B-related BDNF signaling pathway plays an important role in the alleviation of fear generalization.