Preclinical and clinical studies indicate that deficiency in folic acid plays a role in the pathophysiology of depression. Considering that alterations in the signaling pathways that regulate neuroplasticity and cellular survival are implicated in depressive disorders, the present study investigated the involvement of the phosphoinositide 3-kinase (PI3K), glycogen synthase kinase-3 (GSK-3β), and peroxisome proliferator-activated receptor-γ (PPARγ) in the antidepressant-like effect of folic acid in the forced swimming test (FST). The intracerebroventricular (i.c.v.) pre-treatment of mice with LY294002 (10 nmol/site, a PI3K inhibitor) or GW-9662 (1 µg/site, a PPARγ antagonist) prevented the antidepressant-like effect of folic acid (50 mg/kg, p.o.) in the FST. In addition, the administration of subeffective doses of the selective GSK-3β inhibitor, AR-A014418 (3 mg/kg, i.p.), a non-selective GSK-3β inhibitor, lithium chloride (10 mg/kg, p.o) or a PPARγ agonist, rosiglitazone (1 µg/site, i.c.v.) in combination with a subeffective dose of folic acid (10 mg/kg, p.o.) significantly reduced the immobility time in the FST as compared with either drug alone, without altering the locomotor activity. These results indicate that the antidepressant-like effect of folic acid in the FST might be dependent on inhibition of GSK-3β and activation of PPARγ, reinforcing the notion that these are important targets for antidepressant activity.