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Motor imagery training (MIT) has been widely used to improve hemiplegic upper limb function in stroke rehabilitation. The effectiveness of MIT is associated with the functional neuroplasticity of the motor network. Currently, brain activation and connectivity changes related to the motor recovery process after MIT are not well understood.We aimed to investigate the neural mechanisms of MIT in stroke rehabilitation through a longitudinal intervention study design with task-based functional magnetic resonance imaging (fMRI) analysis.We recruited 39 stroke patients with moderate to severe upper limb motor impairment and randomly assigned them to either the MIT or control groups. Patients in the MIT group received 4 weeks of MIT therapy plus conventional rehabilitation, while the control group only received conventional rehabilitation. The assessment of Fugl-Meyer Upper Limb Scale (FM-UL) and Barthel Index (BI), and fMRI scanning using a passive hand movement task were conducted on all patients before and after treatment. The changes in brain activation and functional connectivity (FC) were analyzed. Pearson's correlation analysis was conducted to evaluate the association between neural functional changes and motor improvement.The MIT group achieved higher improvements in FM-UL and BI relative to the control group after the treatment. Passive movement of the affected hand evoked an abnormal bilateral activation pattern in both groups before intervention. A significant Group × Time interaction was found in the contralesional S1 and ipsilesional M1, showing a decrease of activation after intervention specifically in the MIT group, which was negatively correlated with the FM-UL improvement. FC analysis of the ipsilesional M1 displayed the motor network reorganization within the ipsilesional hemisphere, which correlated with the motor score changes.MIT could help decrease the compensatory activation at both hemispheres and reshape the FC within the ipsilesional hemisphere along with functional recovery in stroke patients.