Background: Adrenomedullin (AM), an endogenous peptide, is secreted in response to cerebral ischemia, contributing to neuroprotection by exerting anti-inflammatory and vasodilatory effects. Although previous studies showed elevated levels of mid-regional fragment of pro-AM (MR-proAM), a stable marker of AM, predict unfavorable outcome in acute ischemic stroke (AIS), the role of the biomarker remains to be determined. Methods: Firstly, plasma MR-proAM levels were measured in consecutively enrolled patients with AIS within 4.5 hours of the onset and compared with those of healthy controls. Propensity score was used to match the baseline difference. Secondly, variables associated with the increased MR-proAM levels were evaluated in AIS. Finally, it was determined whether MR-proAM levels are associated with a large penumbra which was defined as ICA/M1 occlusion with NIH stroke scale ≥ 6 and ischemic core volume ≤ 50 ml calculated by the RAPID software. Results: MR-proAM levels in AIS (n = 122; median age, 77 years; male, 59%; median NIHSS 6.5) were higher (median 0.68 vs. 0.42 nmol/L, p < 0.001) compared with controls (n = 1298; median age, 58 years; male, 33%). The difference remained after matching of baseline (p=0.004). In patients with AIS, MR-proAM levels were correlated with NIHSS (r = 0.367, p < 0.001) and eGFR (r = -0.527, p < 0.001). MR-proAM levels did not correlate with ischemic core volume (r = 0.065, p = 0.478), but significantly increased in AIS patients with cardioembolic stroke (CES) (n = 53) (0.80 vs. 0.59 nmol/L, p < 0.001) and ICA/M1 occlusion (n = 40) (0.78 vs. 0.63 nmol/L, p = 0.048). Interestingly, MR-proAM levels were higher (0.85 vs. 0.62, p < 0.001) in AIS patients with a large penumbra (n = 24) compared to those without (n = 98), and the difference remained after adjustment of baseline factors (OR [95% confidence interval], 1.43 [1.10-2.02], p = 0.004). Conclusions: MR-proAM levels increase in patients with AIS, especially in those with large vessel occlusion and clinical imaging mismatch. MR-proAM is a potential biomarker for ischemic penumbra.