Aims More and more advances show that hydrogen sulfide (H2S) sulfhydrates functional proteins and regulates their actions.However, much less is known about the endogenous sulfhydration of key proteins under physiological conditions.Methods Biotinswitch assay, electrophysiological recording, RNA interference, full ESIMS scan et al.Results Herein, we found that the protein sulfhydration was dynamically regulated by neuronal activity.H2S sulfhydrated and activated serine racemase (SR) in the hippocampus, resultantly increased Dserine level.Notably, sulfhydration of SR was markedly promoted by neuronal stimulation and underlay activitydependent changes of Dserine availability.Both genetic knockdown and pharmacological inhibition of cystathionineβsynthase (CBS), the key H2Sproducing enzyme in brain, attenuated hippocampal longterm potentiation(LTP), which can be reversed by exogenous supplement of H2S or Dserine.We also observed that the increase in LTP induced by H2 S is dependent on Dserine and polysulfides.In aged rats, sulfhydration of SR was significantly decreased.Furthermore, exogenous supplement of H2S restored the level of sulfhydration and reversed the agerelated deficits in hippocampal LTP.Conclusion Our data provide direct evidence for the biological significance of endogenous sulfhydration in physiological conditions and demonstrate a pivotal role of activitydependent sulfhydration in synaptic plasticity.