Depression is a potentially life-threatening mental disorder with unknown etiology. Several microRNAs (miRNAs) have been shown to play critical roles in the etiology of depression. Here, we aim to elucidate the anti-depressive behavior of miR-124 suppression in prefrontal cortex (PFC). Quantitative real-time PCR (RT-PCR) was used to evaluate the expression of miR-124 and SIRT1 in the PFC of a chronic unpredictable mild stress (CUMS) model. The PFC of C57BL/6J mice was bilaterally injected with lentiviral vectors (LV) for ectopic expression of SIRT1, miR-124, or miR-124 inhibitor (si-miR-124). The anti-depressive behavior was observed after injection of LV-SIRT1 or LV-si-miR-124 into the PFC, using behavior tests including latency to feed, food and water intake, sucrose preference test, and forced swimming test. MiR-124 overexpression and inhibition resulted in upregulation and down-regulation of SIRT1 and cyclic AMP responsive element binding protein 1 (CREB1), respectively. MiR-124 overexpression exacerbated depression-like behaviors and decreased SIRT1. Further, dual-luciferase assay confirmed that SIRT1 was a target of miR-124. Taken together, a potential molecular regulation of miR-124 on SIRT1 is revealed by our study and miR-124 suppression in PFC is a potential strategy to reduce depression-like behavior.