Aim To study the regulatory effect of acupuncture pretreatment on ferroptosis of nerve cells in rats with ischemic stroke. Methods Sixty SD rats were randomly divided into sham middle cerebral artery occlusion (MCAO) group, MCAO group, and acupuncture+MCAO group. In the acupuncture+MCAO group, the acupuncture points of DU26, PC6, and SP6, were selected for acupuncture pretreatment, once a day for a total of 5 days. After pretreatment, MCAO or sham MCAO models were prepared. The Zausinger sextintegral method was used to score the neurological function of rats, and the infarct volume of brain tissue was calculated by TTC staining. Electron microscopy was used to observe the pathomorphological changes of brain tissue. The iron content was detected by colorimetric method, the content of malondialdehyde (MDA) and glutathione (GSH) was determined by ELISA, and the expression of glutathione peroxidase 4 (GPX4) was detected by immunofluorescence. Results Compared with the sham MCAO group, the MCAO group had a decrease in neurological function scores, a significant increase in infarct volume, a decrease in the number of mitochondria under electron microscopy, a rupture and vacuolization of the inner mitochondrial cristae, an increase in the contents of iron and MDA in brain tissue, and a decrease in GSH content and GPX4 expression. Compared with the MCAO group, the acupuncture+MCAO group had an increase in neurological function scores, a decrease in infarct volume, a large number of mitochondria under electron microscopy, a clear structure, a decrease or disordered arrangement of some mitochondrial crest structures, a decrease in the contents of iron and MDA in brain tissue, and an increase in GSH content and GPX4 expression. Conclusion Acupuncture pretreatment can alleviate neurological damage in rats, and its mechanism may be related to regulating iron, GSH and MDA contents in brain tissue, and GPX4 expression, improving cell antioxidant capacity and inhibiting nerve cell ferroptosis.