Gomisin E increased ACh content and ChAT activity, decreased AChE activity in the hippocampus and ileum, and upregulated the protein expression of CHRM1 and CHRM2 in the hippocampus.<h4>Conclusion</h4>Gomisin E effectively ameliorates D-galactose/PCPA-induced insomnia and the associated depressive-like behaviors and cognitive dysfunction. Here, ACHE is linked to insomnia measurement.