Plant defensins are sulfur-rich cationic peptides, typically characterized by a conserved 3D structure. In general, defensin tertiary structure is formed by a single a-helix followed by three antiparallel b-sheets. These peptides have been isolated from different plants, showing different biological activities as antimicrobial and enzyme inhibitors. Due these facts, the elucidation of defensin mechanism of action is a real challenge and only few hypotheses have been proposed. This work intends to analyze plant defensins that act as a-amylase and proteinase inhibitors by using in silico experiments as molecular modeling and protein docking. For models construction, sequences of Sorghum bicolor and Cassia fistula defensins were submitted to Bioinfo Metaserver in order to find a specific template by threading. FFAS03 and 3D-PSSM indicated that 1GPS, a g-thionin from wheat endosperm, and 1JKZ, a defensin from Pisum sativum, which showed similar structural features to S. bicolor and C. fistula defensins, respectively. Using these templates, models were constructed using MODELLER v8.0. Furthermore, S. bicolor defensin was docked to Tenebrio molitor a-amylase (1TMQ) and C. fistula to bovine trypsin structure by using GRAMM-X v.1.0.1, considering that both act in a canonical fashion style. Gromos 96 was used for energy minimization and Deep View Swiss-Pdb Viewer 3.7 was used to visualization. Complexes were analyzed by Protein-Protein Interaction Server being determined the interface forming residues. Results demonstrated that S. bicolor defensins interacted to TMA probably by ionic interactions between residues Asp₂₈₆ and Glu₂₂₈ from TMA catalytic site and Lys₂₆ and Lys₄₅ from inhibitor, respectively. A hydrogen bond net was also observed, which seems to be important for complex stability. Therefore, C. fistula defensin Lys₀₉ strictly interacts by a loop to trypsin catalytic site by a salt bridge with Asp₁₇₁. These data could help to elucidate the mechanism of action for plant defensins with inhibitory activity and also being useful for generate efficient inhibitors with higher enzyme specificity.