Purpose: Little is known about the function of histone arginine methylation in acute lymphoblastic leukemia (ALL). The objective was to evaluate whether protein arginine methyltransferase 5 (PRMT5) plays a role in pediatric ALL and to determine the possible mechanism of epigenetic regulation. Experimental Design: We used bone marrow samples from patients with pediatric ALL, the Nalm6 cell line, mature B-cell lines, and mouse xenograft models to evaluate the function of PRMT5 in ALL tumorigenesis. Results: This study showed that PRMT5 and the symmetric dimethylation of H4R3 (H4R3sme2) were upregulated in most initially diagnosed (n = 15; 100%) and relapsed (n = 4; 75%) bone marrow leukemia cells from patients with pediatric B-cell precursor ALL (BCP-ALL) and were decreased when the disease was in remission (n = 15; 6.7%). Down-regulation of H4R3sme2 by PRMT5 silencing induced BCP-ALL cell differentiation from the pre-B to immature B stage, whereas overexpressed PRMT5 with enhanced H4R3sme2 promoted human mature B cells to dedifferentiate back to the pre-B II/immature B stages in vitro. High PRMT5 expression enhanced the proportion of CD43(+)/B220(+)/sIgM(-) B leukocytes in recipient mice. CLC and CTSB were identified as potential target genes of PRMT5 in BCP-ALL cells and were inhibited by H4R3sme2 in gene promoters. Conclusions: We demonstrate that enhanced PRMT5 promotes BCP-ALL leukemogenesis partially by the dysregulation of B-cell lineage differentiation. H4R3sme2 and PRMT5 may serve as potential sensitive biomarkers of pediatric BCP-ALL. Suppression of the activation of PRMT5 may offer a promising therapeutic strategy against pediatric BCP-ALL.