Around 280 million people worldwide suffer from depression. However, treatment for this mood disorder is ineffective in 30% of patients. An important target for the treatment of depression is the dorsal raphe nucleus (DRN), which is the main source of serotonin (5-HT) to the forebrain. Previous research has shown that mecamylamine (Mec) increases the activity of 5-HT neurons by enhancing glutamate release through activation of α4β2 nicotinic receptors (nAChRs), while inhibiting GABAergic release by inhibiting α7 nAChRs in the DRN. This mechanism may explain its antidepressant properties. However, there is a paradox regarding how mecamylamine, a non-specific antagonist of nAChRs, activates α4β2 nAChRs. Therefore, the aim of this study was to evaluate the effect of mecamylamine enantiomers on 5-HT neurons and to investigate the role of high and low-sensitivity α4β2 nAChRs in the DRN. We performed electrophysiological recordings in brains slices of male Wistar rats and found that both enantiomers increased the firing rate of 5-HT neurons. Specifically, S-mec increased sEPSCs, whereas R-mec decreased sEPSCs and sIPSCs. The effects of S-mec on 5-HT activity were potentiated by a selective agonist of α4B2 HS nAChRs (TC-2559). These results were supported by a chronic unpredictable stress model. Acute administration of S-mec + TC-2559 reversed the antidepressant phenotype as assessed by the Open Field Test (OFT), Sucrose Preference Test (SPT) and Forced Swim Test (FST). These findings suggest a potential target for the treatment of patients resistant to conventional antidepressant therapies.