〔Abstract〕 Objective To investigate the selective activation of 5-hydroxytryptamine neurons in the dorsal raphe nucleus(DRN5-HT) of rats with insomnia induced by p-chlorophenylalanine(PCPA) using chemical genetics techniques, and to explore whether it has a regulatory effect on comorbidities of insomnia and anxiety. Methods 32 male SD rats were randomly divided into control group, PCPA model group(PCPA group), chemical activation group(hM3Dq+CNO group), and chemical control group(hM3Dq+Saline group). The anxiety level of experimental rats was evaluated through open field experiments and elevated cross maze experiments.In vivoelectroencephalography(EEG) was used to record propofol induced sleep and cortical EEG changes, and immunofluorescence staining was used to observe changes in c-Fos positive protein expression in 5-HT neurons. Results The behavioral results showed that compared with the chemical control group, the anxiety level of the chemical activation group was significantly lower(P<0.05), and sleep duration was significantly prolonged(P<0.05). The expression level of c-Fos positive protein in DRN5-HT neurons in the chemical activation group was higher(P<0.05). The EEG results showed that the percentage of δ-band power between the chemical activation group and the chemical control group was higher(P<0.05), the power percentage in the α-band was relatively low(P<0.05). Conclusion Chemical activation of 5-HT neurons in the DRN region can improve anxiety levels and increase sleep duration in PCPA induced insomnia rats.