AbstractSulfide and persulfide are chemically different and one might expect persulfide to be more effective in mediating sulfur signaling because persulfide can directly modify protein cysteine residue. However, rapid scrambling, and interconversions occur among sulfur species. Then there is the question of whether the chemical reactivity differences between sulfide and persulfide would translate into pharmacological differences. Utilizing a delivery system to generate pure hydrogen sulfide (H₂S), hydrogen persulfide (H₂S₂), and N‐acetyl‐l‐cysteine persulfide (N‐CysSSH), we examined the activities of sulfide and persulfide in vitro and in vivo. Persulfide prodrugs exhibited increased activities compared to the H₂S prodrug. In particular, the H₂S₂ prodrug offers much‐elevated analgesic effects compared to the H₂S prodrug in vivo. Persulfide prodrugs also possess a reduced level of toxicity compared to the H₂S prodrug in vivo, indicating persulfide might represent a better therapeutic paradigm than H₂S.