Voltage-gated calcium channels (VGCCs) participate in many important physiological functions. However whether VGCCs are modulated by changes of osmolarity and involved in anisotonicity-induced nociception is still unknown. For this reason by using whole-cell patch clamp techniques in rat and mouse trigeminal ganglion (TG) neurons we tested the effects of hypo- and hypertonicity on VGCCs. We found that high-voltage-gated calcium current (I(HVA)) was inhibited by both hypo- and hypertonicity. In rat TG neurons, the inhibition by hypotonicity was mimicked by Transient Receptor Potential Vanilloid 4 receptor (TRPV4) activator but hypotonicity did not exhibit inhibition in TRPV4(-/-) mice TG neurons. Concerning the downstream signaling pathways, antagonism of PKG pathway selectively reduced the hypotonicity-induced inhibition, whereas inhibition of PLC- and PI3K-mediated pathways selectively reduced the inhibition produced by hypertonicity. In summary, although the effects of hypo- and hypertonicity show similar phenotype, receptor and intracellular signaling pathways were selective for hypo- versus hypertonicity-induced inhibition of I(HVA).