Voltage-gated potassium channels (VGPCs) play an important role in many physiological functions by controlling the electrical properties and excitability of cells. Changes in tonicity in the peripheral nervous system can activate nociceptors and produce pain. Here, using whole cell patch clamp techniques, we explore how hypo- and hypertonicity modulate VGPCs in cultured rat and mouse trigeminal ganglion (TG) neurons. We found that hypo- and hypertonicity had different effects on slow-inactivating K+ current (IK) and fast-inactivating K+ current (IA): hypotonicity increased IK but had no effect on IA while hypertonicity depressed both IK and IA. The increase of IK by hypotonicity was mimicked by transient receptor potential vanilloid 4 (TRPV4) receptor activator 4alpha-phorbol-12,13-didecanoate (4alpha-PDD) but hypotonicity did not exhibit increase in TRPV4-/- mice TG neurons, suggesting that TRPV4 receptor was involved in hypotonicity-induced response. We also found that inactivation of PKC selectively reversed the increase of IK by hypotonicity, whereas antagonism of G-protein selectively rescued the inhibitions of IK and IA by hypertonicity, indicating that different intracellular signaling pathways were required for the modulation by hypo- and hypertonicity. In summary, changes in osmolality have various effects on IK and IA and different receptors and second messenger systems are selective for the modulation of VGPCs induced by hypo- versus hypertonicity.