Hydrogen sulfide is a signaling molecule that regulates essential processes for plant performance, such as autophagy. In Arabidopsis thaliana, hydrogen sulfide negatively regulates autophagy independently of reactive oxygen species, but the underlying mechanism remains to be understood. To determine whether persulfidation, an emergent posttranslational modification, has a main role in the control of autophagy by sulfide, we have used a proteomic approach targeting ATG4, a cysteine protease that plays a crucial role in autophagy progression. We showed that AtATG4a from A. thaliana, which is the predominant ATG4 protease in this plant species, contains a specific site for persulfidation, the residue Cys170, included in the characteristic catalytic triad Cys-His-Asp of cysteine proteases. We tested whether persulfidation regulates the activity of ATG4 by setting up a heterologous assay using the Chlamydomonas reinhardtii CrATG8 protein as a substrate. Our findings demonstrate that sulfide significantly inactivates AtATG4a cleavage activity in a reversible manner. The biological significance of the reversible inhibition of the ATG4 protease by sulfide is supported by our findings in Arabidopsis leaves under both basal and autophagy-inducing conditions. We also observed a significant increase in the overall ATG4 activity in Arabidopsis under nitrogen starvation and osmotic stress, which is inhibited by sulfide. Therefore, our data strongly suggest that the negative regulation of autophagy by sulfide is mediated, at least, by the specific persulfidation of the protease ATG4.