Modern agriculture allows for the production of foods that are high in n-6 linoleic acid and low in n-3 α-linolenic acid (LAhigh/ALAlow), which are suggested to be associated with an increased risk for the onset of anxiety disorders. However, there is not sufficient evidence to understand its underlying brain mechanism. Given that mouse offspring derived from mothers fed a LAhigh/ALAlow diet during gestation and early lactation showed increased anxiety-related behaviors and that rodents exposed to a LAhigh/ALAlow diet are more vulnerable to stress, in this study, we investigated the effects of maternal LAhigh/ALAlow diet consumption on stress-induced anxiety-related behavior and the brain structures involved in the expression of negative emotional states in mouse offspring. In a standard environment, offspring exposed to either the control diet or the LAhigh/ALAlow diet in utero showed similar stay times in the center zone in the open field test. On the other hand, under stressful environments, offspring exposed to the LAhigh/ALAlow diet in utero showed decreased stay times in the center zone compared to those exposed to the control diet. We further found that the number of a subpopulation of pyramidal neurons in the basolateral amygdala, which can regulate negative emotional behaviors, was greater in the offspring exposed to the LAhigh/ALAlow diet compared to those exposed to the control diet. These data suggest that maternal dietary imbalance between n-6 and n-3 polyunsaturated fatty acids confers stress vulnerability to offspring during the process of brain development.