Pectin methylesterases (PMEs), a multigene family of proteins with multiple differentially regulated isoforms, are key enzymes implicated in the carbohydrates (pectin) metabolism of cell walls. Olive pollen PME has been identified as a new allergen (Ole e 11) of potential relevance in allergy amelioration, since it exhibits high prevalence among atopic patients. In this work, the structural and functional characterization of two olive pollen PME isoforms and their comparison with other PME plants was performed by using different approaches: (1) the physicochemical properties and functional-regulatory motifs characterization, (2) primary sequence analysis, 2D and 3D comparative structural features study, (3) conservation and evolutionary analysis, (4) catalytic activity and regulation based on molecular docking analysis of a homologue PME inhibitor, and (5) B-cell epitopes prediction by sequence and structural based methods and protein-protein interaction tools, while T-cell epitopes by inhibitory concentration and binding score methods. Our results indicate that the structural differences and low conservation of residues, together with differences in physicochemical and posttranslational motifs might be a mechanism for PME isovariants generation, regulation, and differential surface epitopes generation. Olive PMEs perform a processive catalytic mechanism, and a differential molecular interaction with specific PME inhibitor, opening new possibilities for PME activity regulation. Despite the common function of PMEs, differential features found in this study will lead to a better understanding of the structural and functional characterization of plant PMEs and help to improve the component-resolving diagnosis and immunotherapy of olive pollen allergy by epitopes identification.