Low consumption of n-3 polyunsaturated fatty acids (PUFA) during the developmental period has been increasingly associated with an increased risk of depressive-like symptoms in both male and female sexes. Therefore, here we performed behavioral and biochemical quantifications in adolescent rats to evaluate possible sex-driven differences in the development of anxiety-like disorders related to life-long n-3 PUFA low intake. Male and female adolescent rats fed for their entire life with n-3 PUFA poor diet showed an anxiety-like profile compared to n6/n-3 PUFA balanced diet. However, such deficiency led to reduced cortical serotonin (5-HT) in females, while increased GABA levels were retrieved in males. Conversely, in amygdala, 5-HT and noradrenaline (NA) were increased in n-3 PUFA poor treated rats. In male rats, n-3 PUFA poor diet induced significant increase in systemic kynurenine levels, while the pro-oxidant metabolite 3-Hydroxy kynurenine was higher in both sexes. In addition, considering the recent involvement of spleen-brain axis on mood disorders and neuroimmune communication, we evaluated biomarkers in the spleen. N-3 PUFA deprivation reduced NA content and increased the indoleamine 2,3-dioxygenase-1 expression in females, while acetylcholine and tumor necrosis factor alpha were higher in males. Taken together, our data indicated that deficiency of n-3 PUFA in diet induced mood disorders in adolescent animals, however this behavioral phenotype is accompanied by a different immune activation in male and female rats.