Purpose: Little is known about the function of histone arginine methylation in acute lymphoblastic leukemia. The objective was to evaluate whether protein arginine methyltransferase 5 (PRMT5) plays a role in pediatric acute lymphoblastic leukemia and to determine the possible mechanism of epigenetic regulation. Experimental Design: We used bone marrow (BM) samples from pediatric acute lymphoblastic leukemia (ALL) patients, 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%) BM leukemia cells from pediatric B-cell precursor ALL (BCP-ALL) patients and were decreased when the disease was in remission (n=15; 6.7%). Downregulation 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 and its downstream targets may offer a promising therapeutic strategy against pediatric BCP-ALL.