Hydrogen sulfide (H₂S) is an endogenous gasotransmitter implicated in the regulation of protein function. This study investigates whether H₂S-derived sulfur species can engage the catalytic Zn²⁺ center of histone deacetylase 6 (HDAC6) and influence HDAC6-dependent deacetylation. Molecular dynamics simulations support accessibility of HS⁻ to the HDAC6 CD2 active-site tunnel, whereas biochemical, biophysical and quantum-chemical analyses support a zinc-dependent interaction and the chemical feasibility of Zn–sulfur coordination. To investigate the relationship between endogenous H₂S production and HDAC6-dependent protein acetylation, acetylomic profiling was performed in EA.hy926 cells with knockout of cystathionine γ-lyase (CSE, encoded by CTH) or HDAC6. This dataset contains the acetylomics data comparing CSE-knockout versus wild-type cells and HDAC6-knockout versus wild-type cells.tunnel to identify the catalytic zinc ion yielding an S-Zn bond to promote enzyme activity. Since HDAC6 plays a pivotal role in regulating the acetylation levels of non-histone proteins, this project was designed to explore effects of endogenous hydrogen sulfide generating enzyme CSE and HDAC6 knockout on the acetylomics of EA.hy 926 cells.