Abstract Body: Background and Purpose: Aged patients experience more cognitive dysfunction than young patients after stroke. Brain astrocytes and microglia causes excessive removal of synapses at the early stage of stroke. Inhibition of their phagocytosis improved neurobehavioral outcomes. Long-term post-stroke cognitive dysfunction in aged subjects may be associated with increased synapse pruning by astrocytes, as increased reactive astrocytes are present in and around the atrophic region. Hypothesis: Excessive synapse pruning by reactive astrocytes contributes to the long-lasting post-stroke memory dysfunction in aged mice. Methods: pMCAO was induced in young (2-month-old) and aged (15-18-month-old) mice. Memory performance was tested weekly for 8 weeks by Y-maze, and at 8 weeks post-stroke by novel objective recognition (NOR) tests. Brains were collected 8 weeks after pMCAO. Gene expressions were analyzed by RNAseq and western blot. Atrophic volume, CD68⁺ cells, GFAP⁺ cells, and synaptophysin (SYP) were analyzed histologically. Results: In Y-maze test, aged stroke mice made fewer spontaneous alternations from 3 to 8 weeks after pMCAO than young stroke and sham operated aged mice. In NOR test, aged stroke mice spent shorter time on the novel objects than young stroke and sham aged mice. RNAseq data showed up-regulation of inflammation, and down-regulation of axon growth and synaptic transmission pathways in the aged ipsilateral than young ipsilateral cortex and aged contralateral cortex. Glutamatergic and cholinergic synapses were decreased in aged ipsilateral cortex and hippocampus. GABAergic presynapse protein was increased in the aged ipsilateral hippocampus compared to the young mice. All support reduced activity in the cortex and hippocampus of aged stroke mice. Aged mice showed larger atrophic volumes, more CD68⁺ and GFAP⁺ cells in the peri-atrophic and hippocampi regions than young mice. About 10-fold more GFAP⁺ cells were detected in aged peri-atrophic and ipsilateral hippocampi regions than CD68⁺ cells; 57% GFAP⁺ and 37% CD68⁺ cells were SYP⁺ in the ipsilateral hippocampi, 53% GFAP⁺ and 39% CD68⁺ cells were SYP⁺ in the peri atrophic region of aged stroke brain, indicating that reactive astrocytes contributed more than microglia on synapse pruning in aged mice. Conclusions: Reactive astrocytes contribute more than microglia to synapse pruning at the chronic stage of stroke, which is involved in long-lasting post-stroke memory dysfunction in the aged mice.