Abstract <p>Hydrogen sulfide has been recognized as an effective endogenous signaling gasotransmitter, and exhibits antiproliferative activity against several cancer cells. To improve the antitumor activity of ergosterol peroxide (EP), a series of ergosterol peroxide hydrogen sulfide donor derivatives were synthesized by conjugating EP with an H₂S-donating compound ADT-OH [5-(4-hydroxyphenyl)-3<i>H</i>-1,2-dithiole-3-thione]. Most derivatives showed more potent antiproliferative activities than EP against A549 and HepG2 cell lines, while they were lack of sensitivity to MCF-7 cells. In particular, (3β,5α,8α,22<i>E</i>)-5,8-epidioxyergosta-6,22-dien-3-yl 5-[4-(3-thioxo-3<i>H</i>-1,2-dithiol-5-yl)phenyl] 3,3-dimethylpentanedioate showed the most potent antiproliferative activities against A549 cells with IC₅₀ value of 2.52 μM, which demonstrated 7.4-fold improvement comparing to the parent compound EP. In the H₂S release experiment, (3β,5α,8α,22<i>E</i>)-5,8-epidioxyergosta-6,22-dien-3-yl 5-[4-(3-thioxo-3<i>H</i>-1,2-dithiol-5-yl)phenyl] 3,3-dimethylpentanedioate was able to release a greater amount of H₂S and reached its maximum value within 15 min. (3β,5α,8α,22<i>E</i>)-5,8-Epidioxyergosta-6,22-dien-3-yl 5-[4-(3-thioxo-3<i>H</i>-1,2-dithiol-5-yl)phenyl] 3,3-dimethylpentanedioate was also found have a strong binding affinity (–9.1 kcal/mol) with ATP-binding pocket of epidermal growth factor receptor (EGFR) protein. Additionally, the results of ADMET predictions suggested that these compounds possess favorable drug-like properties and good membrane permeability. The above findings indicate that this compound may become a highly promising lead compound for the development of anti-non-small cell lung cancer drugs.</p>