The first and probably most important bond is between a mother and her infant promoting their psychological and physiological well-being. Here, the “pro-maternal” oxytocin system as well as the “anti-maternal” CRF system are strongly involved in the formation and maintenance of this maternal bond. Losing a child is a severely stressful experience that dramatically affects the maternal brain, and those neuropeptide systems are promising candidates concerning the underlying emotional and behavioral consequences. Our study endeavored to investigate the changes in rat mothers' CRF system activity after one day of mothering experience followed by permanent bond disruption for 19 days compared to undisturbed mothers. We assessed their passive stress-coping behavior in the forced swim test without pharmacological CRF-R treatment. We further determined mRNA expression of CRF, CRF-R 1 and CRF-R 2 by qPCR in different brain regions involved in maternal behavior and emotionality, i.e., paraventricular nucleus (PVN), amygdala (AMY), and ventromedial hypothalamus (VMH). Permanent pup separation led to increased passive stress-coping in the mothers, and this could be reversed by acute central CRF-R blockade. Furthermore, mRNA expression of CRF-R 1 in PVN and AMY and of CRF-R 2 in VMH increased following pup loss; no other changes were detected. We conclude that region-specific alteration in CRF system activity might underly the emotional consequences in rat mothers following pup loss. Our studies present a first approach to advance our understanding how permanent separation from the offspring can cause a dysregulation of the brain CRF system thereby leading to severe emotional consequences.