Previously, anti-inflammatory properties of 3,4,5-Trihydroxycinnamic acid (THC) has been reported in lipopolysaccharide (LPS)-induced RAW264.7 murine macrophage cells and in an LPS-induced sepsis BALB/c mice animal model. However, the effect of THC on the anti-allergic effect in mast cells has not been elucidated. The current study aimed to demonstrate the anti-allergic properties of THC and its underlying mechanism. Rat basophilic leukemia (RBL-2H3) cells were treated with phorbol-12-myristate-13-acetate (PMA) and A23187, a calcium ionophore, to be activated. The anti-allergic effect of THC was determined by measuring cytokine and histamine release. Western blotting was conducted to determine mitogen-activated protein kinases (MAPKs) activation and nuclear factor-κB (NF-κB) translocation. THC significantly suppressed PMA/A23187-induced tumor necrosis factor α secretion and THC also significantly attenuated degranulation, releasing β-hexosaminidase and histamine in concentration-dependent manners. Furthermore, THC significantly attenuated PMA/A23187-induced cyclooxygenase 2 expression and nuclear translocation of NF-κB. THC significantly suppressed PMA/A23187-induced increased phosphorylation of p38 mitogen-activated protein kinase, phosphorylated (p-)extracellular signal-regulated kinase 1/2 and p-c-Jun N-terminal kinase in RBL-2H3 cells. Overall, the results demonstrated that THC exhibited anti-allergic action by significantly attenuating degranulation of mast cells through the inhibition of MAPKs/NF-κB signaling pathway in RBL-2H3 cells.