TWIK-related spinal cord K+ (TRESK) channel is abundantly expressed in trigeminal ganglion (TG) and dorsal root ganglion neurons and is one of the major background K+ channels in primary afferent neurons. Mutations in TRESK channels are associated with familial and sporadic migraine. In rats, both chronic nerve injury and inflammation alter the expression level of TRESK mRNA. Functional studies indicate that reduction of endogenous TRESK channel activity results in hyper-excitation of primary afferent neurons, suggesting that TRESK is a potential target for the development of new analgesics. However, whether and how enhancing TRESK channel activity would decrease the excitability of primary afferent neurons has not been directly tested. Here, we over-expressed TRESK subunits in cultured mouse TG neurons by lipofectamine-mediated transfection and investigated how this altered the membrane properties and the excitability of the small-diameter TG population. To account for the heterogeneity of neurons, we further divided small TG neurons into two groups, based on their ability to bind to fluorescently-labeled isolectin B (IB4). The transfected TG neurons showed a 2-fold increase in the level of TRESK proteins. This was accompanied by a significant increase in the fraction of lamotrigine-sensitive persistent K+ currents as well as the size of total background K+ currents. Consequently, both IB4-positive and IB4-negative TG neurons over-expressing TRESK subunits exhibited a lower input resistance and a 2-fold increase in the current threshold for action potential initiation. IB4-negative TG neurons over-expressing TRESK subunits also showed a significant reduction of the spike frequency in response to supra-threshold stimuli. Importantly, an increase in TRESK channel activity effectively inhibited capsaicin-evoked spikes in TG neurons. Taken together, our results suggest that potent and specific TRESK channel openers likely would reduce the excitability of primary afferent neurons and therefore are potential therapeutics for the treatment of migraine and other chronic pain symptoms.