Background: Spider venoms contain peptide toxins that modify ion channel currents, primarily in excitable insect cells. Research has revealed various peptides with pharmacological properties affecting mammalian species, including immune cell ion channels like the voltage-gated proton channel Hv1. This channel facilitates reactive oxygen species (ROS) production during the respiratory burst, contributing to inflammation and carcinogenesis by maintaining an acidic tumor microenvironment that supports tumor growth, invasion, and resistance to apoptosis. Experimental Approach: In this study, six animal venoms underwent screening against hHv1 using the patch clamp technique. The complete venom extracted from the spider Grammostola rosea resulted in the modulation of hHv1 expressed in CHO cells during whole-cell patch clamp measurements. HPLC reverse-phase fractionation of the venom showed that two peptide fractions #7 (GsAF-I) and #9 (GsAF-II) were active . Key Results: GsAF-I and GsAF-II inhibited Hv1 with micromolar Kd values in a membrane potential-dependent manner. Voltage ramp protocol revealed a shift in the Hv1 activation threshold toward a more positive potential. Voltage step protocol showed slowed activation kinetics and accelerated deactivation kinetics, suggesting stabilization of the closed state. The effects of both peptides were reversible. The membrane potential dependence of the inhibition was uniquely analysed and presented in this study. Conclusion and Implications: This is the first study to investigate in detail the inhibitory effect of peptides from animal venoms on the human Hv1 ion channel. The found two peptides effectively modify proton currents, highlighting their potential as lead compounds for drug development.