Mast cell-mediated reactions promote various allergic disease, including anaphylaxis, allergic rhinitis, asthma, and atopic dermatitis. Different data demonstrated an intricate relationship between the use of antihistaminic drugs, the onset of side effects, and the development of resistance, underscoring the importance to find novel therapeutic approaches to treat allergic diseases. Olive leaf extract (OLE), is a by-product of the olive tree rich in bioactive compounds, known for its numerous therapeutic properties, including antioxidant, anti-tumoral and antidiabetic effects. In this study, we investigated the effect of OLE on the mast-cell-mediated allergic inflammation using human mast cells HMC-1.2. OLE reduced histamine and β-Hexosaminidase release from HMC cells activated by phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI) through modulation of calcium signal. Moreover, OLE decreased the PMACI-stimulated gene expression of proinflammatory cytokines such as tumor necrosis factor-a (TNF-α), interleukin-8 (IL-8) and interleukin-6 (IL-6) in human mast cells. This result was confirmed by multiplex assay in which the pre-treatment with OLE reduced the effective secretion of TNF-α, IL-6 and IL-8. These effects were correlated to ROS reduction and modulation of both mitochondrial mass and membrane potential. Finally, the inhibitory effect of OLE was nuclear factor (NF)-kB dependent as demonstrated by both activity assay and Western Blot analysis. Taken together, our results demonstrated that OLE inhibits mast-cell-derived allergic inflammation modulating mast cells degranulation, proinflammatory cytokines release and NF-kB activation. Therefore, OLE could represent a novel potential therapeutic approach for the treatment of mast cell–associated disorders.