Insomnia is a common sleep disorder worldwide, and oxidative stress and mitochondrial damage are closely related to insomnia. This study aimed to investigate the mechanism by which senegenin exerts neuroprotective effects in regulating oxidative stress and mitochondrial damage in insomnia. In vivo, EEG/EMG analysis confirmed the successful establishment of insomnia rat models; Nissl and HE staining and electron microscopy were used to evaluate the pathological changes of neurons and mitochondria in rat brain tissue. The expression of oxidative stress and sleep factors was assessed. In vitro, an oxidative damage cell model was established to measure oxidative stress-related parameters; the protective concentration of senegenin against oxidative damage was determined using the CCK-8 assay, and the effects of senegenin on the expression of Keap1/Nrf2 and PINK1/Parkin, key signaling pathways involved in oxidative stress and mitochondrial damage, were analyzed. During insomnia, wake is prolonged, and NREM and REM are shortened; learning memory and exploration behavior are impaired, oxidative stress factor expression is changed, and mitochondria are damaged. Brain tissue from insomnia rats showed decreased BDNF, 5-HT1A, GABA-T, and GAD and increased expression of 5-HT2A and Glu. Keap1, PINK1, Parkin, and LC3 expression increased and Nrf2, NQO1, HO-1, and p62 expression decreased in oxidatively injured cells. Senegenin showed a dose-response regulatory effect after the intervention. Senegenin may exert neuroprotective effects in insomnia by improving oxidative stress and mitochondrial damage.