Impaired awareness of hypoglycaemia (IAH) is characterised by reduced behavioural and endocrine counter-regulatory responses (CRR); deficits which develop following repeated hypoglycaemia (RH). These impairments lead patients to experience recurrent severe hypoglycaemic episodes, with associated morbidity and mortality. Treatments remain ineffective and our mechanistic understanding is incomplete. Corticotrophin-releasing hormone (CRH) neurons, in the paraventricular nucleus of the hypothalamus (PVH), modulate responses to physiological stressors, including a single hypoglycaemia (SH) episode. How CRH neurons respond to RH remains unclear. Eight-week old male Crh-cre::EYFP mice were randomised to SH (n = 6), RH (n = 6) or saline (n = 5) groups. RH mice received repeated injections with insulin (1.75 U/kg, subcutaneous) three times a week for four weeks. In parallel, SH and saline groups received repeated injections of 0.9% NaCl. Blood glucose was recorded using tail-tip sampling. On the final injection day, SH and RH groups received insulin. 2 hours after injection, mice were anaesthetised with isoflurane (3-4 ml min-1) and perfused transcardially with 4% paraformaldehyde. 30μm coronal brain sections were used to label EYFP and cFos immunoreactive neurons. Data (mean±SEM) were analysed using one-way ANOVA with Tukey’s post-hoc comparisons, with significance set at p <0.05 . Insulin administration led to a consistent reduction in blood glucose, with a nadir of 3.8 ± 0.4 mmol/l on the final day of injection. In RH mice, the duration of recovery to baseline blood glucose, was observed to increase with successive hypoglycaemic episodes. SH and RH significantly increased cFos in the PVH compared with saline (565 ± 37 vs 279 ± 31 cells, p<0.005; 432 ± 53 vs 279 ± 31 cells, p<0.05). Within the mid-PVH (bregma -0.82 to -0.94mm AP), which contained the greatest concentration of labelled cells, significantly higher cFos expression was noted with SH than RH (322 ± 15 vs 215 ± 32 cells/section, p<0.05). The medial parvocellular dorsal division (mpd) of the PVH contained the greatest density of CRH neurons (150 ± 12 cells/section). CRHmpdPVH neurons are highly responsive to SH, with 73 ± 5% of neurons expressing cFos. Co-localisation of cFos in CRHmpdPVH neurons, was significantly higher for SH compared with RH (103 ± 15 vs 51 ± 10 cells/section, p<0.05). We conclude that CRHPVH neurons, particularly in the mpdPVH, are activated by hypoglycaemia. However, with RH, these neurons become less responsive. These findings suggest that RH, akin to other forms of repeated stress, leads to adaptive changes within stress-responsive neurons. Further understanding of the role of CRHPVH neurons within the central glucose-regulatory network and their response to RH, may explain the CRR deficits which arise in patients with IAH.