Use of the inhalation anesthetic isoflurane may increase the risk of cognitive deficiency and neurotoxicity after birth. A growing body of evidence suggests that melatonin is an effective treatment for various types of oxidative stress damage and neurodegenerative disease. In this study, we aimed to examine the effects of melatonin on isoflurane-induced endoplasmic reticulum (ER) stress, spatial learning and memory impairment during devel-opment. The rats were grouped according to whether the rats were exposed to isoflurane or a control gas and whether they were administered melatonin or phosphate buffered saline (PBS). We administered isoflurane to 7 -day-old Sprague-Dawley rat pups with intraperitoneal injections of melatonin (20 mg/kg) 15 min before and 3 h after the initiation of anesthesia. Twelve hours after isoflurane anesthesia, rats were randomly selected from each group and sacrificed. The hippocampal tissue and serum were collected to determine the levels of SIRT1, Mfn2, PERK, and other proteins or cytokines related to ER stress, apoptosis, and neuroinflammation. Subsequently, all remaining rats were assessed for spatial learning and memory deficiency 31 days after birth using the Morris water maze test. We found that melatonin attenuated isoflurane-induced ER stress and neuroapoptosis in the hippocampus and decreased the level of neuroinflammatory markers in the serum of newborn rats, resulting in improved spatial learning and memory. In addition, the neuroprotective effect of melatonin was weakened after the SIRT1/Mfn2/PERK signaling pathway was suppressed by lentivirus transfection. Therefore, our findings demonstrate that melatonin ameliorates spatial learning and memory impairment after isoflurane exposure, and these beneficial effects are associated with a reduction in ER stress, neuroapoptosis, and neuroinflammation via the SIRT1/Mfn2/PERK signaling pathway.