Mercury sulfide (HgS) exerts extensive biological effects on neuronal function. To investigate the direct target of HgS in neuronal cells, we developed a biotin-tagged HgS probe (bio-HgS) and employed an affinity purification technique to capture its target proteins. Then, we identified S-phase kinase-associated protein 1 (Skp1) as a potential target of HgS. Unexpectedly, we discovered that HgS covalently binds to Skp1 through a "Cys62-HgS-Cys120" mode. Moreover, our findings revealed that HgS inhibits the ubiquitin-protease system through Skp1 to up-regulate SNAP-25 expression, thereby triggering synaptic vesicle exocytosis to regulate locomotion ability in C. elegans. Collectively, our findings may promote a comprehensive interpretation of the pharmacological mechanism of mercury sulfide on neuroprotective function.