Increased n-6 and reduced n-3 long-chain PUFA (LC-PUFA) intake in Western diets may contribute to the increased prevalence of allergic diseases. Key effector cells in allergy are mast cells (MC). The aim of the present study was to investigate the effects of n-6 v.n-3 LC-PUFA on MC phenotype. Human MC lines (LAD2 and HMC-1) were incubated for 24 h with either arachidonic acid (AA, n-6 LC-PUFA) or the n-3 LC-PUFA EPA or DHA. The effects of these three LC-PUFA on degranulation, mediator secretion and reactive oxygen species (ROS) generation were assessed. ROS, mitogen-activated protein kinase (MAPK) or NF-κB inhibitors were used to unravel signalling pathways involved in cytokine secretion. AA, EPA or DHA did not reduce IgE-mediated degranulation by LAD2 cells. However, AA increased PGD2 and TNF-α secretion by ionomycin/phorbol 12-myristate 13-acetate-stimulated HMC-1, whereas EPA and DHA more prominently inhibited IL-4 and IL-13 secretion. Suppression of IL-4 and IL-13 release by LC-PUFA correlated with reduced ROS generation. IL-4 and IL-13 release by activated HMC-1 was abrogated using ROS inhibitors. Inhibition of MAPK signalling, but not NF-κB, downstream of ROS reduced IL-13 secretion by activated HMC-1. Combined incubation of EPA or DHA with MAPK inhibitors further suppressed IL-13 secretion. In conclusion, the n-6 LC-PUFA AA enhanced pro-inflammatory mediator production by MC, while the n-3 LC-PUFA EPA as well as DHA more effectively suppressed ROS generation and IL-4 and IL-13 release. This suggests that dietary supplementation with EPA and/or DHA may alter the MC phenotype, contributing to a reduced susceptibility to develop and sustain allergic disease.