Pediatric high-grade gliomas (PHGG) are aggressive, undifferentiated central nervous system tumors with poor outcomes, for which no standard-of-care drug therapy currently exists. Through a knockdown (KD) screen for epigenetic regulators, we identified PRMT5 as essential for PHGG cell growth. We hypothesized that, similar to its effect in normal cells, PRMT5 promotes self-renewal of stem-like PHGG tumor-initiating cells essential for tumor growth. We conducted in vitro analyses, including limiting dilution studies of self-renewal, to determine the phenotypic effects of PRMT5 KD. We performed chromatin immunoprecipitation sequencing (ChIP-Seq) to identify PRMT5-mediated epigenetic changes and performed gene set enrichment analysis to identify pathways that PRMT5 regulates. Using an orthotopic xenograft model of PHGG, we tracked survival and histologic characteristics resulting from PRMT5 KD or administration of a PRMT5 inhibitor ± radiation therapy. In vitro, PRMT5 KD slowed cell-cycle progression, tumor growth and self-renewal, and altered chromatin occupancy at genes associated with differentiation, tumor formation, and growth. In vivo, PRMT5 KD increased survival and reduced tumor aggressiveness; however, pharmacologic inhibition of PRMT5 with or without radiation therapy did not improve survival. PRMT5 KD epigenetically reduced tumor-initiating cells' self-renewal, leading to increased survival in preclinical models. Pharmacologic inhibition of PRMT5 enzymatic activity may have failed in vivo due to insufficient reduction of PRMT5 activity by chemical inhibition, or this failure may suggest that nonenzymatic activities of PRMT5 are more relevant. Implications: PRMT5 maintains and promotes the growth of stem-like cells that initiate and drive tumorigenesis in PHGG.