BACKGROUND: Ershiwuwei Shanhu pills (ESWWSHW, Tibetan name is བྱུ་དམར་ཉེར་ལྔ།) is a traditional Tibetan medicinal formulation utilized for the management of cerebral ischemia, which mainly includes 25 medicinal materials, including 14 plant medicines and 11 mineral medicines. In the eighth century AD, ESWWSHW was created by Yutuo Yuandan Gongbu and later included in the classic Tibetan medical work Four Medical Classics. At present, it was included in the Ch.P (2020 edition, volume 1). ESWWSHW can open up orifices, unblock meridians, and relieve pain. It is primarily utilized for treating white nerve disease (Tibetan medicine disease name, including cerebral ischemia), confusion, brain pain, epilepsy, and various neuropathic pains. At present, the mechanism underlying ESWWSHW resistance to cerebral ischemic injury remains unknown. STUDY DESIGN AND METHODS: In this research, the chemical constituents present in ESWWSHW and blood-infiltrated tissues were subjected to qualitative analysis using UPLC-Q-TOF-MS, while the chemical constituents within the preparations were quantitatively assessed. 27 chemical constituents from 12 batches of ESWWSHW were subjected to content determination by HPLC-QqQ-MS. Sprague-Dawley (SD) rats were randomly divided into the sham group; model group; nimodipine group; and ESWWSHW low-dose, ESWWSHW medium-dose, and ESWWSHW high-dose groups. The rat model of cerebral ischemia reperfusion was established by utilizing a suture technique. The pharmacodynamic indices employed in this study included the cerebral infarction ratio and neurobehavioral ratings. Using metabolomics and protein chip technology, a amount of 14 differential metabolites were identified, predominantly associated with the sphingolipid and glycerophospholipid metabolic pathways, which exhibit properties related to antioxidative stress and the inhibition of apoptosis. In addition, the pathological examinations of the diencephalon, cortex, and hippocampus; TUNEL staining; and molecular biology (PCR, WB, and IF) were used to verify the mechanism by which ESWWSHW regulates endoplasmic reticulum stress-mediated apoptosis to resist cerebral ischemic injury. RESULTS: In the prescription of ESWWSHW, a amount of 76 chemical constituents were identified, with nine of which were compared with reference standards. Following a 24-hour period of cerebral ischemia in rat subjects, seven medicinal herb prototype components were qualitatively identified as entering the bloodstream, and 49 medicinal herb prototype components were identified as entering tissues. Metabolomic analysis revealed 14 metabolites that exhibit differential expression, which are implicated in the metabolic pathways of sphingolipids and glycerophospholipids. Additionally, protein chip technology identified 18 proteins with differential expression. Notably, inflammatory cytokines such as IL-10, IL-3, and IL-7 may serve as critical targets for interventions aimed at mitigating cerebral ischemic injury. ESWWSHW show to ameliorate neurobehavioral abnormalities in rats subjected to the MCAO model, reduce the incidence of cerebral infarction, mitigates pathological changes associated with neuronal necrosis in the cortical regions of brain tissue, enhances the quantity of Nissl bodies in these areas, and suppresses the expression of apoptotic cells within the cortical regions. ESWWSHW significantly elevates the mRNA expression level of ATF6, XBP1, and BCL2 in ischemic brain tissue, while concurrently decreasing the protein expression levels of CHOP, GRP78, BAX, and NLRP3, and increasing the protein expression of XBP1. CONCLUSION: The protective mechanism of ESWWSHW against cerebral ischemic injury may be associated with the modulate cellular apoptosis induced by ERS-induced, particularly via the regulation of the GRP78/XBP1/CHOP signaling pathway. This suggests significant potential for its application as a therapeutic strategy in the management of cerebral ischemic injury.