This study aimed to assess the effects of human urinary kallidinogenase (HUK) on motor function outcome and corticospinal tract recovery in patients with acute ischemic stroke (AIS). This study was a randomized, controlled, single-blinded trial. Eighty AIS patients were split into two groups: the HUK and control groups. The HUK group was administered HUK and standard treatment, while the control group received standard treatment only. At admission and discharge, the National Institutes of Health Stroke Scale (NIHSS), Barthel Index (BI) and muscle strength were scored. The primary endpoint was the short-term outcomes of AIS patients under different treatments. The secondary endpoint was the degree of corticospinal tract fiber damage under different treatments. There was a significant improvement in the NIHSS Scale, BI and muscle strength scores in the HUK group compared with controls (Mann-Whitney U test; P < 0.05). Diffusion tensor tractography classification and intracranial arterial stenosis were independent predictors of short-term recovery by linear regression analysis. The changes in fractional anisotropy (FA) and apparent diffusion coefficient (ADC) decline rate were significantly smaller in the HUK group than in the control group (P < 0.05). Vascular endothelial growth factor (VEGF) increased significantly after HUK treatment (P < 0.05), and the VEGF change was negatively correlated with changes in ADC. HUK is beneficial for the outcome in AIS patients especially in motor function recovery. It may have protective effects on the corticospinal tract which is reflected by the reduction in the FA and ADC decline rates and increased VEGF expression. The study was registered on ClinicalTrials.gov (unique identifier: NCT04102956).Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.