Long-term exposure to stress plays a key role in the pathogenesis of major depression. Recently, the ventrolateral orbital cortex (VLO) has received considerable attention for its role in the antidepressant response. However, the mechanisms underlying stress response in the VLO remain largely elusive. MiR-101 has been implicated in regulating multiple neurological processes. The present study used the chronic unpredictable mild stress (CUMS) rat model to investigate the expression of miR-101 in the VLOs of rat brains and the possible relevance of miR-101 to depression. Furthermore, an intra-VLO administration of a miR-101 mimic was performed to provide insights into the miR-101-mediated dysregulation mechanisms associated with depression. The results showed that chronic stress induced typical depressive-like behaviors in rats and decreased miR-101 levels in the VLOs of rat brains. Moreover, the dual specificity phosphatase 1 (DUSP1) protein levels were increased in the VLOs in CUMS rats, whereas the ERK phosphorylation and BDNF levels in the CUMS rats were decreased. Enhancing miR-101 expression via an intro-VLO microinjection of its mimic reversed the depressive-like behaviors in CUMS rats. Intra-VLO treatment with the miR-101 mimic also attenuated the upregulation of CUMS-induced DUSP1 expression and inhibited the downstream ERK phosphorylation and BDNF expression. These results suggest that miR-101 has functional significance in the pathophysiology of stress-induced dysfunctions in the VLO. MiR-101 may directly regulate DUSP1 expression, and the mechanism underlying the antidepressant effects of miR-101 may involve the negative regulation of DUSP1 expression, which in turn promotes downstream ERK/BDNF signaling.