This study was conducted to examine the effects of weaning stress on gene expression of specific markers in hypothalamus-pituitary-adrenal (HPA) axis and neuronal activity in the newly weaned piglets. Twelve 28-days-old, newly weaned crossbred (Landrace x Yorkshire x Duroc) male piglets from 6 l (2 piglets/l) were randomly categorized into two groups: (a) weaning stress: piglets were separated from their dams, relocated and mixed with the unacquainted domestic piglets for 2 hr (stress, n = 6); (b) no-stress: piglets stayed with their dams in the farrowing house (NS; n = 6). After weaning stress, all piglets were electrically euthanized and the blood samples/HPA tissues were collected for subsequent analysis, including plasma cortisol and mRNA expression of c-fos, c-jun, corticotropin-releasing hormone (CRH), CRH receptor 1 (CRHR-1) and adrenocorticotropin hormone receptor (MC2R). Results: Weaning stress significantly (p < 0.05) increased the plasma cortisol level and suppressed the expression of c-fos and CRH in hypothalamus. In addition, weaning stress enhanced the mRNA abundance of c-jun and CRHR-1 in the pituitary gland. No significant differences in the gene expression of MC2R and CRHR-1 were observed in the adrenal gland between treatment groups. Taken together, HPA involved in weaning stress and CRHR-1 and c-jun could be potential markers to evaluate the activation of HPA axis.