The gasotransmitter hydrogen sulfide (H2S) is implicated in a myriad of biological processes, including disease causing alterations. Besides acting as an antioxidant molecule, H2S reacts with thiol groups from cysteine residues found in proteins. This post-translational modification (PTM), called S-persulfidation, plays an essential role during endoplasmic reticulum (ER) stress. Here we demonstrated that yeast cystathionine beta-synthase (Cys4), is the main responsible for ER H2S-mediated homeostasis. We also found that during ER stress, Cys4 interacts with specific proteins that we identified with at least one cysteine residue modified by hydrogen sulfide. Finally, we focused on one interactor with reported tunicamycin sensitivity, the phospholipid transfer protein Sec14. In addition to Cys4 immunoprecipitation, we confirmed this interaction by pulling down Sec14 interactors. Deleting CYS4 generated the accumulation of lipid droplets inside the cytosol, similar to Sec14. For the first time, we described a set of protein-protein interactions of Cys4 during ER stress with cysteines susceptible to S-persulfidation, leaving an open question about the role and regulation of the rest of the interactors. SIGNIFICANCE: In this study, we identified several interactor partners of yeast cystathionine beta synthase and analyzed cysteine residues with cysteine S-persulfidations. Although yeast have multiple H2S producing enzymes, Cys4 seems to play a predominant role during ER stress. Temporal and spatial generation of H2S is becoming significant in the field, and our findings contribute to that understanding. Besides, Cys4/Sec14 complex found here, seven different Cys4 complexes that may be regulated by H2S were also reported. Because the biogenesis of this PMT is still controversial, findings like these support the hypothesis of interaction mediated biogenesis. H2S producing enzymes specificity would help control their signaling across the cell. Finally, all Cys4 interactors with modified cysteines, including Sec14, are excellent targets to study how this PTM regulates enzyme functioning. Structural and functional assays with cysteine mutants in these proteins will shed light on new regulatory mechanisms.