ABSTRACT Objectives This study aimed to longitudinally observe the improvement mechanism of semantic fluency in subacute post-stroke aphasia (PSA) patients using resting-state functional magnetic resonance imaging (rs-fMRI). Methods Twelve PSA patients, about one month after onset, were enrolled in this study and received speech-language therapy (SLT) for one month. Auditory comprehension and semantic fluency were evaluated using the Western Aphasia Battery (WAB) and the Animal Fluency Test. Before and after treatment, rs-fMRI data were collected, and the dice similarity coefficient was used to measure the spatial similarity between each patient’s lesion and a reference lesion. The left posterior inferior temporal gyrus (pITG) was used as a seed to calculate the normalized functional connectivity in whole-brain voxel analysis using DPABI software for statistical analysis. Results The dice similarity coefficient between each patient’s lesion and the reference lesion showed moderate to high intensity (0.57 ± 0.14) in the Montreal Neurological Institute space. After treatment, we found a significant increase in functional connectivity between the left pITG and the right prefrontal lobe convergence area (peak t = 8.219, Gaussian random field multiple comparison correction, voxel p < 0.001, cluster p < 0.05). The increase in functional connectivity was negatively correlated with the improvement in auditory comprehension (r =–0.707, p = 0.033) and positively correlated with the improvement in semantic fluency (r = 0.79, p = 0.02). Conclusion The improvement of semantic fluency in subacute PSA patients may require the participation of the right convergence area of the prefrontal lobe.