Stressful early life events can have short- and long-term effects on neuroendocrine and behavioural mechanisms of adaptation. Here, we investigated the effects of a single social isolation (4 h) of domestic piglets on both behavioural alterations in open-field tests and modifications in the expression of genes regulating glucocorticoid response in stress-related brain regions at 7, 21 or 35 days of age. The mRNAs of glucocorticoid receptor (GR), mineralocorticoid receptor (MR), 11ss-hydroxysteroid dehydrogenase 1 and 2 (11ss-HSD1 and 11ss-HSD2) and c-fos were analysed by real-time RT-PCR in the hypothalamus, hippocampus and amygdala. The social isolation caused both elevated stress hormone concentrations (e.g. cortisol) and open-field reactivity (e.g. locomotion, vocalisation) compared to control piglets. The enhanced behavioural and neuroendocrine activity was associated with distinct changes in gene expression in the limbic system. The hypothalamic GR, MR and 11ss-HSD1 mRNA expressions and the hippocampal 11ss-HSD1 mRNA was significantly higher in isolated piglets, whereas in the amygdala social isolation caused a significant decrease in MR mRNA expression. Isolated piglets also displayed significantly higher c-fos mRNA expression, an estimate of neuronal activation, in hypothalamus and amygdala. The mRNA alterations as well as the behavioural and hormonal pattern show an effect of social isolation on days 7 and 21, but no effect on day 35. In conclusion, a single social isolation in piglets caused age-dependent neuroendocrine and behavioural changes that indicate increased arousal and experienced distress. The present results also suggest that psychosocial stress effects should be considered for the assessment of livestock handling practices with respect to health and welfare.