Therapeutic hypothermia and histone deacetylase inhibitors, such as valproic acid (VPA), independently have been shown to have neuroprotective properties in models of cerebral ischemic and traumatic brain injury. However, the depth of hypothermia and the dose of VPA needed to achieve the desired result are logistically challenging. It remains unknown whether these two promising strategies can be combined to yield synergistic results. We designed an experiment to answer this question by subjecting hippocampal-derived HT22 cells to severe hypoxia in vitro. Mouse hippocampal HT22 cells were exposed to 200 μM cobalt chloride (CoCl(2)), which created hypoxic conditions in vitro. Cells were incubated for 6 or 30 hours under the following conditions: (1) Dulbecco's Modified Eagle Medium; (2) 200 μM CoCl(2); (3) 200 μM CoCl(2) plus 1 mmol/L VPA; (4) 200 μM CoCl(2) plus 32°C hypothermia; and (5) 200 μM CoCl(2) plus both 1 mmol/L VPA and 32°C hypothermia. Cellular viability was evaluated by (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and lactate dehydrogenase release assays at 30 hours after treatment. Levels of acetylated histone H3, hypoxia-inducible factor-1α, phospho-GSK-3β, β-catenin, and high-mobility group box-1 were measured by Western blotting. High levels of acetylated histone H3 were detected in the VPA-treated cells. The release of lactate dehydrogenase was greatly suppressed after the combined hypothermia + VPA treatment (0.269 ± 0.003) versus VPA (0.836 ± 0.026) or hypothermia (0.451 ± 0.005) treatments alone (n = 3, P = .0001). (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay showed that the number of viable cells was increased by 17.6 % when VPA and hypothermia were used in combination (n = 5, P = .0001). Hypoxia-inducible factor-1α and phospho-GSK-3β expression were synergistically affected by the combination treatment, whereas high-mobility group box-1 was increased by VPA treatment, and inhibited by the hypothermia. This is the first study to demonstrate that the neuroprotective effects of VPA and hypothermia are synergistic. This novel approach can be used to develop more effective therapies for the prevention of neuronal death.