Much evidence from animal and clinical studies has shown that the mediodorsal nucleus of the thalamus (MD) is related to various types of memory, such as visual recognition, object-reward association, spatial working, and reference memory; however, few studies have investigated its role in emotion-related learning and memory processes. This study compared the effect of pre- and posttraining bilateral lesions of the mediodorsal thalamic nucleus with those of the amygdala on contextual conditioned fear. Both pre- and posttraining amygdala lesions almost eliminated conditioned freezing, and significantly blocked postshock freezing when behavioral tests were performed immediately after footshocks, reconfirming previous studies that the amygdala is implicated in the learning of Pavlovian conditioning. Both pre- and posttraining lesions of the mediodorsal nucleus of the thalamus significantly attenuated conditioned freezing but had no effect on postshock freezing. In contrast to lesions of the amygdala, those of the mediodorsal thalamic nucleus failed to alter the increased defecation induced by conditioned fear stress. Our results suggest that the mediodorsal nucleus of the thalamus has an important role in acquisition, consolidation or retrieval in Pavlovian contextual fear conditioning. Possible neural circuits, incorporating the amygdala, MD, and hippocampus, and the functional similarity of the MD and hippocampus in contextual fear conditioning, are also discussed.