To select the appropriate site of transfection of the collapsin response mediator protein 2 (CRMP2) eukaryotic expression plasmid in the cortex of rat brains. Methods: A total of 150 adult male Sprague-Dawley rats were randomly divided into groups vacant plasmid (VP), lateral ventricle (LV), hippocampus (H), cortex (C), and olfactory bulb (OB). After the appropriate site were chosen, 20 rats were divided into groups sham, middle cerebral artery occlusion (MCAO), (MCAO+GFP), (MCAO+CRMP2/GFP). The CRMP2 eukaryotic expression plasmid and vacant plasmid were respectively injected into the four different parts of MCAO/reperfusion model rats by stereotactic surgery; 24 h and 48 h after transfection, expression of CRMP2 protein and mRNA were tested by Western blot and RT-PCR assay and the expression of GFP was examined by laser scanning confocal microscope. TTC staining was employed to observe the cerebral infarct volume, and neurological function was also evaluated. Results: After injection of the plasmid, CRMP2 expression levels in the cortex increased; the expression levels in the LV and H groups were significantly higher than those in the C and OB groups (P<0.05) while there was no statistical difference between the LV and H groups (P>0.05). Compared to MCAO without plasmid treatment, CRMP2 eukaryotic plasmid intervention reduced cerebral infarction volume and promoted neural functional recovery (P<0.05). Conclusion: The CRMP2 eukaryotic expression plasmid can successfully transfect rat cortical tissue and lead to increased expression of CRMP2 proteins and mRNA in the ischemic cortical regions. The transfection rate is higher in the lateral ventricle than in the other three locations.