Lactating rats must continuously maintain a critical balance between caring for pups and aggressively responding to nest threats. We tested the neural response of lactating females to the presentation of their own pups and novel intruder males using blood oxygen level-dependent functional magnetic resonance imaging at 7 T. Dams were presented with a single sequence of a control stimulus, pups or a male intruder in one imaging session (n = 7-9). To further determine the selectivity of neural processing, dams were imaged for their response to a male intruder in both the absence and presence of their pups (n = 6). Several maternal cortical and limbic brain regions were significantly activated by intruder presentation but not by pups or a control stimulus. These included the nucleus accumbens, periaqueductal gray, anterior cingulate, anterior thalamus, basal nucleus of the amygdala, temporal cortex, prelimbic/orbital area and insula. The nucleus accumbens, periaqueductal gray, temporal cortex and mediodorsal thalamus still showed greater neural activity when compared with intruder presentation in the absence of pups. The present results suggest that the high emotional state generated by a threat to pups involves robust activation of classical limbic regions controlling emotional responses. This pattern of blood oxygen level-dependent activity may precede behavioral states upon which lactating rats initiate attacks against a potential threat to offspring.