Objectives To evaluate the diagnostic yield and safety of brainstem stereotactic biopsy for brainstem lesions. Methods We performed a meta-analysis of English articles retrieved from the PubMed, Web of Science, Cochrane Library, and APA psycInfo databases up to May 12, 2021. A binary fixed-effect model, the inverse variance method, or a binary random-effect model, the Dersimonian Laird method, were utilized for pooling the data. This meta-analysis was registered with INPLASY, INPLASY202190034. Findings A total of 41 eligible studies with 2792 participants were included. The weighted average diagnostic yield was 97.0% (95% confidential interval [CI], 96.0-97.9%). The weighted average proportions of temporary complications, permanent deficits, and deaths were 6.2% (95% CI, 4.5–7.9%), .5% (95% CI, .2–.8%), and .3% (95% CI, .1–.5%), respectively. The subgroup analysis indicated a nearly identical weighted average diagnostic yield between MRI-guided stereotactic biopsy and CT-guided stereotactic biopsy (95.9% vs 95.8%) but slightly increased proportions of temporary complications (7.9% vs 6.0%), permanent deficits (1.9% vs .2%), and deaths (1.1% vs .4%) in the former compared to the latter. Moreover, a greater weighted average diagnostic yield (99.2% vs 97.6%) and lower proportions of temporary complications (5.1% vs 6.8%) and deaths (.7% vs 1.5%) were shown in the pediatric patient population than in the adult patient population. Conclusions Brainstem stereotactic biopsy demonstrates striking accuracy plus satisfying safety in the diagnosis of brainstem lesions. The diagnostic yield, morbidity, and mortality mildly vary based on the diversity of assistant techniques and subject populations.