BACKGROUND: Thrombolytic therapy remains the standard treatment for acute ischemic stroke. The narrow window for thrombolysis means that delay in treatment worsens brain injury. Astrocytes regulate blood-brain barrier (BBB) integrity and neuroinflammation, yet their neurotoxic polarization exacerbates injury in neuropathological conditions, including ischemic stroke. Delayed recombinant tissue plasminogen activator (rt-PA) thrombolysis disrupts astrocyte homeostasis, further aggravating brain injury. This study investigates whether acupuncture, a key therapy in traditional Chinese medicine, alleviates delayed thrombolysis-induced injury by modulating astrocyte neurotoxic polarization and elucidates the underlying signaling pathway. METHODS: A rat model of embolic stroke with delayed rt-PA thrombolysis was established. The effects of infarct volume, BBB integrity, and neuroinflammation of acupuncture were evaluated. Bulk and single-cell transcriptomic analyses were performed to assess astrocyte-specific transcriptional changes. Western blotting, immunofluorescence, and pharmacological inhibition experiments validated molecular mechanisms. RESULTS: Acupuncture reduced infarct volume, improved neurological function, and restored BBB integrity. Transcriptomic analysis revealed dynamic regulation of astrocyte neurotoxic polarization following acupuncture intervention. Further validation experiments demonstrated that acupuncture suppressed the ERK1/2-Cx43 cascade, thereby attenuating astrocyte-mediated neurotoxicity. Pharmacological modulation of this pathway replicated the protective effects of acupuncture, highlighting the role in mitigating astrocyte dysfunction and promoting BBB recovery. CONCLUSION: Acupuncture mitigates delayed thrombolysis-induced brain injury by modulating astrocyte polarization via the ERK1/2-Cx43 pathway. These findings highlight acupuncture as a potential strategy to enhance thrombolytic therapy safety in ischemic stroke.