Barley (Hordeum vulgare) is a widely cultivated cereal crops, and its production is increasingly threatened by environmental stresses such as drought and salinity. Hydrogen sulfide is established as a signaling molecule that promotes tolerance to plant stress throught persulfidation, a post-translational modification of cysteine residues in proteins. The purpose of this study is to explore the impact of NaHS (sulfide donor) pretreatment on barley plants in enhancing tolerance to drought and salinity stresses, and determine if persulfidation is involved. In pretreated-plants, phenotypical traits and pigment contents showed an improvement in the survival of the plants under stress conditions. Quantification of stress-markers such as anthocyanin, proline, and reactive oxygen species also showed significant decreased contents in pretreated compared to untreated plants. In addition, the accumulation of amino acids under drought stress was significantly reduced when plants were pretreated with NaHS. Similarly, the increase of ABA content as a typical drought response was reduced in the pretreated plants. When plants are exposed to salt stress, the Na+/K+ ratio was maintained low in NaHS-pretreated plants, by increasing K+ levels. The sulfide ameliorative effect to salt was also observed during germination in previously NaHS-soaked seeds. Our findings suggest that sulfide pretreatment prepares barely plants to better deal with drought and salinity. Moreover, persulfidation was analyzed under all conditions, exhibiting enhanced levels under stress when plants were pretreated with NaHS. Our findings indicate that sulfide pretreatment induces a previous state in barley to respond more efficiently to stress and propose persulfidation is the underlying mechanism.