Plasma in several organisms has components that promote resistance to envenomation by inhibiting specific proteins from snake venoms, such as phospholipases A2 (PLA2s). The major hypothesis for inhibitor’s presence would be the protection against self-envenomation in venomous snakes, but the occurrence of inhibitors in non-venomous snakes and other animals has opened new perspectives for this molecule. Thus, this study showed for the first time the structural and functional characterization of the PLA2 inhibitor from the Boa constrictor serum (BoaγPLI), a non-venomous snake that dwells extensively the Brazilian territory. Therefore, the inhibitor was isolated from B. constrictor serum, with 0.63% of recovery. SDS-PAGE showed a band at ~25 kDa under reducing conditions and ~20 kDa under non-reducing conditions. Chromatographic analyses showed the presence of oligomers formed by BoaγPLI. Primary structure of BoaγPLI suggested an estimated molecular mass of 22 kDa. When BoaγPLI was incubated with Asp-49 and Lys-49 PLA2 there was no severe change in its dichroism spectrum, suggesting a non-covalent interaction. The enzymatic assay showed a dose-dependent inhibition, up to 48.2%, when BoaγPLI was incubated with Asp-49 PLA2, since Lys-49 PLA2 has a lack of enzymatic activity. The edematogenic and myotoxic effects of PLA2s were also inhibited by BoaγPLI. In summary, the present work provides new insights into inhibitors from non-venomous snakes, which possess PLIs in their plasma, although the contact with venom is unlikely.