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Background: Both hydrogen sulphide (H2S) and mild hypothermia have been reported to prevent brain damage caused by reperfusion assault through regulating the N-methyl-D-aspartate receptor (NMDAR). However, the relationship between the two treatments and how they exert neuro-protective effects through NMDARs remain to be elucidated. Methods: Transient cerebral ischemia was induced using the Pulsinelli four-vessel occlusion method. We used sodium hydrosulphide (NaHS) as the H2S donor. We randomly divided 100 Sprague-Dawley rats into five groups of 20: Sham operation group (Sh), normothermic (36-37 °C) ischemia group (NT), mild hypothermic (32-33 °C) ischemia group (mHT), normothermic ischemia combined with NaHS treatment group (NT + NaHS), and mild hypothermic ischemia combined with NaHS treatment group (mHT + NaHS). After 6 hrs of reperfusion, rats were decapitated and hippocampus samples were immediately collected. We measured NR2A (GluN1), NR2B (GluN2) and p-CREB protein levels using western blotting. We further analyzed BDNF mRNA expression by real-time PCR. Hematoxylin and eosin (HE) staining was used to examine pyramidal cell histology at the CA1 region. All statistical analyses were carried out by ANOVA and LSD t-test as implemented by the SPSS 13.0 software. Results: In the four test groups with ischemia-reperfusion, hippocampal H2S concentration increased following treatment, and administration of NaHS further increased H2S levels. Moreover, administration of both NaHS and mild hypothermia resulted in up-regulation of NR2A and NR2B protein expressions, as well as p-CREB protein and BDNF mRNA levels. At the cellular level, NaHS and mild hypothermia groups exhibited lower damage caused by ischemia-reperfusion in the CA1 region of the hippocampus. The strongest protective effect was observed in rats treated with combined NaHS and mild hypothermia, suggesting their effects were additive. Conclusion: Our results support previous findings that hydrogen sulphide and mild hypothermia can prevent ischemia-reperfusion injury. Both treatments caused an up-regulation of NMDA receptors, as well as an elevation in p-CREB protein and BDNF mRNA levels. Thus, hydrogen sulphide and mild hypothermia may provide neuro-protective effect through activating the pro-survival CREB signaling pathway. [ABSTRACT FROM AUTHOR]