Hypothermia is a promising neuroprotective strategy to improve functional outcome after stroke. Cytokines are produced shortly after the insult and contribute to the resulting damage. Therefore, this study investigated the effect of mild hypothermia on pro- and anti-inflammatory cytokines after a transient focal cerebral ischemia. Endothelin-1 (Et-1), a vasoconstrictor, was infused in the vicinity of the middle cerebral artery to elicit a transient focal cerebral ischemia in male Wistar rats. Rats subjected to two hours of mild hypothermia (34°C) starting 20 minutes after Et-1 injection, were compared to a normothermic group (37°C). The parameters assessed were functional outcome (neurological deficit score), infarct volume and the concentrations of inflammatory (Interleukin-1 beta, Tumor Necrosis Factor alpha) and anti-inflammatory (Transforming Growth Factor beta) cytokines at different time points after stroke. Hypothermia significantly reduced the infarct volume compared to normothermia. This reduction correlated with behaviour as hypothermic rats showed a better outcome after the insult. However, even in normothermic rats, neurological deficit improved in time. Stroke induced an increase in pro-inflammatory cytokines in the core and the penumbra that peaks at 24 hours after the insult. Application of hypothermia lead to increased levels of Tumor Necrosis Factor alpha at 8 hours after the insult and decreased levels of the pro-inflammatory cytokines at 24 hours. In contrast, hypothermia had no effect on the stroke induced increase in Transforming Growth Factor beta. These results suggest that beneficial effects of hypothermia on infarct volume and functional outcome may be, at least in part, mediated by modulation of pro-inflammatory cytokine expression.