BACKGROUND: The clinical application of recombinant tissue plasminogen activator (rt-PA) is significantly constrained by hemorrhagic transformation (HT), a common and severe complication following thrombolysis for ischemic stroke. Notably, the mitochondrial injury-mediated NLRP3 inflammasome plays a crucial role in HT after delayed rt-PA thrombolysis in acute ischemic stroke. Although acupuncture has demonstrated antioxidant and anti-inflammatory effects in acute cerebral infarction, its impact on delayed rt-PA thrombolysis, especially concerning mitophagy and the NLRP3 inflammasome, remains unclear. This study investigates how acupuncture protects against HT resulting from mitochondrial damage and NLRP3 inflammasome activation after delayed rt-PA thrombolysis in acute cerebral stroke. METHODS: We selected an embolic stroke model in rats and assessed brain injury after delayed rt-PA in acute ischemic stroke using neurological deficit score, volume of brain infarct, the permeability assay of the blood-brain barrier (BBB), and HT. Then, the levels of proteins and mRNA involved in mitophagy and the NLRP3 inflammasome pathway were measured by western blot and real-time PCR. The levels of interleukin-18 (IL-18) and interleukin-1β (IL-1β) were assessed using enzyme-linked immunosorbent assay (ELISA). Morphological changes in the BBB and mitochondria of neurons were observed via transmission electron microscopy. RESULTS: Acupuncture significantly improved neurological deficit scores, volume of cerebral infarction, BBB destruction, and HT in an embolic stroke model rat. Furthermore, acupuncture induced mitophagy and substantially downregulated the activity of the NLRP3 inflammasome. Additionally, the use of mitochondrial inhibitors significantly reversed the suppressive impact of acupuncture on the NLRP3 inflammasome. CONCLUSION: Acupuncture can promote mitophagy and suppress NLRP3 inflammasome activation to decrease HT after delayed rt-PA therapy for acute ischemic stroke.