Ara-C is one of the antineoplastic drugs, immune suppressants and a pyrimidine nucleoside. Though Ara-C is an important drug in the treatment of acute myeloid leukemia (AML), there are encouraged consequences about the therapeutic capability of Ara-C in acute lymphoblastic leukemia (ALL). Herein, we evaluated the effect of Ara-C on the expression of genes coding for the enzymes DNA methyltransferase (DNMT) 3A, DNMT 3B and histone deacetylase 3 (HDAC3) in the human B cell-ALL cell line Nalm6. Moreover, we investigated its effects on Nalm6 cells proliferation and apoptosis. Briefly, Nalm6 cells and also normal peripheral blood mononuclear cells (PBMCs) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, and treated with Ara-C at their exponential growth phase. Cell apoptosis rates were studied using Annexin-V/PI staining and fluorescence-activated cell sorting (FACS) analysis, and their proliferation levels were evaluated upon treatment with increasing concentration of Ara-C (5–80 nM) by MTT assay. Finally, the expressions of the above-mentioned 3 genes were quantified using real-time PCR. Based on analysis, Ara-C could powerfully trigger apoptosis and obstructs proliferation of Nalm6 cells upon treatment. After Ara-C treatment, expressions of the genes DNMT3A in Nalm6 cell line increased but DNMT3B and HDAC3 decreased significantly compared with the control group. Altered expressions of the above-mentioned genes in ALL cells under the effect of Ara-C suggests that epigenetic changes such as DNA hypermethylation and histone deacetylation may be appropriate goals in the development of new therapies.