Prolyl carboxypeptidase (PRCP) is an enzyme associated with cerebrovascular risk factors such as hypertension, diabetes mellitus, obesity and hyperlipidemia. We aim to evaluate the relation between serum PRCP activity and severity, evolution and outcome of acute ischemic stroke. We used a specific RP-HPLC activity assay to measure PRCP activity in serum of 50 stroke patients at admission, and at 24 h, 72 h and 7 days after stroke onset to assess correlations with stroke severity based on the National Institutes of Health Stroke scale score (NIHSS), infarct volume on brain MRI scan, stroke outcome based on the modified Rankin scale (mRS) and mortality at 3 months after stroke. The average PRCP activity in serum decreased significantly the first 24 h after stroke onset and returned to baseline values at day 7. High NIHSS scores and infarct volumes at admission were related with a more pronounced decrease of PRCP in the first 24 h after stroke (ΔPRCP24, r = 0.31, P < 0.05; r = 0.30, P < 0.05). In addition, patients who displayed a more pronounced decrease in PRCP levels during the first 24 h after stroke were more likely to be institutionalized upon discharge (n = 21) (ΔPRCP24 ± SD, 0.05 ± 0.10 U/L vs. 0.17 ± 0.14 U/L, P = 0.001). The decrease in PRCP levels in the first 24 h after stroke onset is associated with stroke severity and an unfavourable short-term stroke outcome.