Objective To identify the most consistent and replicable characteristics of altered spontaneous brain activity in mesial temporal lobe epilepsy patients with unilateral hippocampal sclerosis (MTLE-HS). Methods A systematic literature search was performed in PubMed, Embase, The Cochrane Library, China National Knowledge Infrastructure, Wanfang, and CQVIP databases, to identify eligible whole-brain resting state functional magnetic resonance imaging studies that had measured differences in amplitude of low-frequency fluctuations or fractional amplitude of low-frequency fluctuations between patients with MTLE-HS and healthy controls from January 2000 to January 2019. After literature screening and data extraction, Anisotropic Effect-Size Signed Differential Mapping software was used for voxel based pooled meta-analysis. Results Nine datasets from six studies were finally included, which contained 207 MTLE-HS patients and 239 healthy controls. The results demonstrated that, compared with the healthy controls, the MTLE-HS patients showed increased spontaneous brain activity in right hippocampus and parahippocampal gyrus, right superior temporal gyrus, left cingulate gyrus, right fusiform gyrus, and right inferior temporal gyrus; while decreased spontaneous brain activity in left superior frontal gyrus, right angular gyrus, right middle frontal gyrus, left inferior parietal lobule, left precuneus, and right cerebellum (P<0.005, cluster extent≥10). Conclusion The current meta-analysis demonstrates that patients with MTLE-HS show increased spontaneous brain activity in lateral and mesial temporal regions and decreased spontaneous brain activity in default mode network, which preliminarily clarifies the characteristics of altered spontaneous brain activity in patients with MTLE-HS.