Objective: To explore the different compensatory mechanisms of brain function between the patients with brain dysfunction after acute ischemic stroke (AIS) in dominant hemisphere and the non-dominant hemisphere, based on Resting-state Functional Magnetic Resonance Imaging (Rs-fMRI).Methods: In this trial, 15 healthy subjects(HS) were used as blank controls. 30 hemiplegic patients with middle cerebral artery acute infarction of the different dominant hemisphere were divided into the dominant hemisphere group (DH) and the non-dominant hemisphere group (NDH), scanned by a 3.0 T MRI scanner, to obtain the amplitude of low frequency fluctuations (ALFF) and regional homogeneity (ReHo) and compare the differences.Results: Compared with the HS, increased ALFF values in the brain areas such as bilateral midbrain were observed in DH. While decreased ReHo values in the brain areas such as right postcentral gyrus (BA3) were also observed. Enhanced ALFF values in the brain areas such as left BA6, and enhanced ReHo values in the brain areas such as left precuneus were observed in NDH. And the ALFF and ReHo values of right BA9 and precentral gyrus were both increased. Compared with DH, NDH have lower ALFF values in left supplementary motor area and lower ReHo values in right BA10.Conclusion: After acute infarction in the middle cerebral artery of the dominant hemisphere, a compensation mechanism is triggered in brain areas of the ipsilateral cortex regulating motor-related pathways, while some brain areas related to cognition, sensation and motor in the contralateral cortex are suppressed, and the connection with the peripheral brain regions is weakened. After acute infarction in the middle cerebral artery of the non-dominant hemisphere, compensatory activation appears in motor control-related brain areas of the dominant hemisphere. After acute middle cerebral artery infarction in the dominant hemisphere, compared with the non-dominant hemisphere, functional specificity in bilateral supplementary motor area weakens. After acute middle cerebral artery infarction in different hemispheres, there are hemispheric differences in the compensatory mechanism of brain function. Trial registration: Ethics Committee of the China-Japan Union Hospital at Jilin University approval. Trial Registration Number:Chinese Clinical Trial Registry ChiCTR-IOR-15007672. Registered July 18, 2016 (No. 2016ks043).