The high-affinity receptor for immunoglobulin E (IgE) (FcεRI)-mediated activation of mast cells plays an important role in various allergic diseases. To assess the anti-allergic activity of natural vanadium-containing Jeju groundwater (JW), an in vivo passive cutaneous anaphylaxis (PCA) animal model and in vitro mouse bone marrow-derived mast cells (BMMCs) was used. JW inhibited cyclooxygenase-2 (COX-2)-dependent prostaglandin D₂ (PGD₂) generation in a dose-dependent manner, with a concomitant reduction of COX-2 protein expression in IgE-induced BMMCs. In addition, JW inhibited 5-lipoxygenase (5-LOX)-dependent generation of leukotriene C₄ (LTC₄) as well as degranulation in a dose-dependent manner. These results demonstrate that JW has dual COX-2/5-LOX inhibitory activity. In addition, vanadium pentoxide (V₂O₅), which is the major vanadium component of JW, also inhibited PGD₂ and LTC₄ generation as well as degranulation in IgE-induced BMMCs. Furthermore, oral administration of JW dose-dependently inhibited mast cell-dependent passive anaphylactic reaction in IgE-sensitized mice. Taken together, these results suggest that JW may be useful in regulating mast cell-mediated allergic response through the suppression of eicosanoid generation and degranulation in mast cells.