The detailed knowledge about the contribution of immunoproteasome to the neuroinflammation in ischemic stroke is still not available. The immunoreactivity of low molecular mass peptide 2 (LMP2) and low molecular mass peptide 7 (LMP7) was evident in the ipsilateral ischemic cerebral cortex and striatum following transient middle cerebral artery occlusion (MCAO). Both LMP2 and LMP7 increased as early as 4 h after the MCAO, further increased at 24 h, peaked at 72 h and decreased 7 days later. LMP2 and LMP7 were mainly present in astrocytes and microglia/macrophage cells, respectively. LMP2 knockdown by shRNA (short hairpin RNA) markedly reduced the levels of LMP2 and LMP7 protein and caused 75.5 and 78.6% decrease in the caspase-like (C-L) and chymotrypsin-like (CT-L) activities, respectively. Compared with cont-shRNA group (39.7%, infarction volumes/total ipsilateral hemisphere), the infarction volumes were reduced to 22.5% in LMP2-shRNA group. Additionally, LMP2 knockdown significantly reduced activated astrocytes and microglia, the expression nuclear factor kappa B (Nf-kB) p65, tumor necrosis factor-α (TNf-α) and interleukin-1β (IL-1β) and caused less accumulation of ischemia-induced protein ubiquitination compared with MG132. These findings demonstrate that inhibition of LMP2 significantly attenuates inflammatory reaction and offers neuroprotection against focal cerebral ischemia in rats, suggesting that selective immunoproteasome inhibitors may be a promising strategy for stroke treatment.