Redox reactions of cysteine residues lead to a range of reversible post-translational modifications of proteins, regulating their functions. Ca2+-independent phospholipase A2γ (iPLA2γ) belongs to a family of enzymes which catalyze the hydrolysis of membrane glycerophospholipids to liberate free fatty acids and lysophospholipids, contributing to cellular signaling, membrane homeostasis and energy metabolism. The activity of iPLA2γ is regulated by a redox-sensitive process, suggesting a thiol-based modification. Using recent computational methods, we searched the primary sequence of human iPLA2γ for potential cysteine residues that could be involved in a thiol-based redox regulation of the protein. Two cysteines, Cys233 and Cys329, were pointed out as highly probable S-sulfenylation target sites. In addition, we used protein structure homology modeling to create a three-dimensional structure of iPLA2γ to visualize the topology of the predicted cysteine residues. The primary sequence was aligned to search for suitable templates and the model was optimized by energy minimization using the GROMOS96 parameter set and checked for errors in the 3D protein structure. The final 3D structure with visualized cysteine residues was designed using molecular graphic system PyMOL. Using isolated recombinant human iPLA2γ, both Cys233Ser and Cys329Ser targeted mutations revealed altered regulation of the catalytic activity by H2O2. These results are consistent with the participation of iPLA2γ in cellular redox homeostasis and serve as preliminary tools leading to a direct identification of cysteine oxidative modifications of the enzyme. Supported by grant GA15-02051S to M.J. from the Grant Agency of the Czech Republic.