<p>Ketamine is recognized for its rapid-onset and longer-term antidepressant actions, but the molecular mechanisms underlying these outcomes are not fully comprehended. Our prior research indicated that the covalent modification of ketamine or its metabolites on hippocampus protein may contribute to its antidepressant actions, however, the specific molecular mechanisms are yet to be elucidated. In this research, we employed chemical proteomics approaches to investigate comprehensively the covalent interactions between ketamine or its metabolites and hippocampus proteins in vivo. We discovered that ketamine could covalently bind to lysine residues on proteins after bioactivation, complementing our previous finding of cysteine modification. Moreover, comprehensive chemical proteomics analysis revealed that 21 proteins were modified by ketamine or its metabolites in mouse hippocampus. Finally, bioinformatics analysis revealed that ketamine exerted antidepressant effects via multi-target and multi-pathway mechanism especially involved in synaptic plasticity. These findings offer a novel perspective for understanding the underlying molecular mechanism of antidepressant action.</p>