Fast and sustained antidepressant effects of ketamine identified the mammalian target of rapamycin (mTOR) signaling pathway as the main modulator of its antidepressive effects. Thus, mTOR signaling has become integral for the preclinical evaluation of novel compounds to treat depression. However, causality between mTOR and depression has yet to be determined. To address this, we knocked down mTOR expression in mice using an acute intracerebral infusion of small interfering RNAs (siRNA) in the infralimbic (IL) or prelimbic (PrL) cortices of the medial prefrontal cortex (mPFC), and evaluated depressive- and anxious-like behaviors. mTOR knockdown in IL, but not PrL, cortex produced a robust depressive-like phenotype in mice, as assessed in the forced swimming test (FST) and the tail suspension test (TST). This phenotype was associated with significant reductions of mTOR mRNA and protein levels 48 h post-infusion. In parallel, decreased brain-derived neurotrophic factor (BDNF) expression was found bilaterally in both IL and PrL cortices along with a dysregulation of serotonin (5-HT) and glutamate (Glu) release in the dorsal raphe nucleus (DRN). Overall, our results demonstrate causality between mTOR expression in the IL cortex and depressive-like behaviors, but not in anxiety.