Exposure to early life stress causes increased stress responsiveness and permanent changes in the central nervous system. We recently showed that delayed gastric emptying (GE) and accelerated colonic transit (CT) in response to acute restraint stress (ARS) were completely restored following chronic homotypic stress (CHS) in rats via upregulation of hypothalamic oxytocin (OXT) expression. However, it is unknown whether early life stress affects hypothalamic OXT circuits and gastrointestinal motor function. Neonatal rats were subjected to maternal separation (MS) for 180 min/day for 2 wk. Anxiety-like behaviors were evaluated by the elevated-plus-maze test. GE and CT were measured under nonstressed (NS), ARS, and CHS conditions. Expression of corticotropin-releasing factor (CRF) and OXT in the paraventricular nucleus (PVN) of the hypothalamus was evaluated by real time RT-PCR and immunohistochemistry. MS increased anxiety-like behaviors. ARS delayed GE and accelerated CT in control and MS rats. After CHS, delayed GE and accelerated CT were restored in control, but not MS, rats. CRF mRNA expression was significantly increased in response to ARS in control and MS rats. Increased CRF mRNA expression was still observed following CHS in MS, but not control, rats. In response to CHS, OXT mRNA expression was significantly increased in control, but not MS, rats. The number of OXT-immunoreactive cells was increased following CHS in the magnocellular part of the PVN in control, but not MS, rats. MS impairs the adaptation response of gastrointestinal motility following CHS. The mechanism of the impaired adaptation involves downregulation of OXT and upregulation of CRF in the hypothalamus in MS rats.