We have previously demonstrated that arginine-rich and poly-arginine peptides possess potent neuroprotective properties, with poly-arginine peptide R18 identified as being highly effective at reducing infarct volume following middle cerebral artery occlusion (MCAO) in the Sprague Dawley rat. Since peptides synthesised using D-isoform amino acids have greater stability than L-isoform peptides due to increased resistance to proteolytic degradation, they represent potentially more effective peptide therapeutics. Therefore we compared the neuroprotective efficacy of R18 and its D-enantiomer R18D following permanent MCAO in the Wistar rat. Furthermore, as increased peptide stability may also increase peptide toxicity, we examined the effects of R18 and R18D on cultured cortical neurons, astrocytes, brain endothelial cells (bEND.3), and embryonic kidney cells (HEK293) following a 10-minute or 24-hour peptide exposure duration. The in vivo studies demonstrated that R18D resulted in a greater reduction in mean infarct volume compared to R18 (33%, p = 0.004 vs 12%, p = 0.27) after intravenous administration at 300 nmol/kg 30 minutes after MCAO. Both R18D and R18 reduced cerebral hemisphere swelling to a comparable degree (27%, p = 0.03 and 30%, p = 0.02), and improved neurological assessment scores (1.5, p = 0.02 and 2, p = 0.058 vs 3 for vehicle). No abnormal histological findings specific to peptide treatments were observed in hematoxylin and eosin stained sections of kidney, liver, spleen, lung and heart. In vitro studies demonstrated that R18 and R18D were most toxic to neurons, followed by astrocytes, HEK293 and bEND.3 cells, but only at high concentrations and/or following 24-hour exposure. These findings further highlight the neuroprotective properties of poly-arginine peptides, and indicate that R18D at the dose examined is more potent than R18 in Wistar rats, and justify continued investigation of the R18 peptide as a novel neuroprotective agent for stroke.