Background: Low expression of E2F3a and caspase 8 associated protein 2 (CASP8AP2) are associated with poor prognosis of childhood acute lymphoblastic leukemia (ALL). Methods: Dual-luciferase reporter assay and wild type as well as four mutated types of reporter plasmids were used to demonstrate the activation of E2F3a on CASP8AP2 transcription. The direct binding of E2F3a with the promoter of CASP8AP2 was shown by Chromatin Immunoprecipitation (ChIP). Cell proliferation activity and cell cycle were determined by MTS and flow cytometry in leukemic cells after treating with common chemotherapeutic drugs vincristine and daunorubicin. Results: In this study, we found that up-regulation of E2F3a in leukemic cells led to increased fraction of cells in S and G2/M phase, accelerated proliferation, and enhanced sensitivity to vincristine and daunorubicin. ChIP and luciferase assay indicated that E2F3a could directly bind to two fragments in the wild type of CASP8AP2 promotor (-206 to -69 and -677 to -507), and activate its transcription activity which was reduced in mutated promotors. The effect of E2F3a on chemotherapeutic sensitivity of leukemic cells could be reversed by down-regulating CASP8AP2. Conclusions: E2F3a could promote transcription and expression of CASP8AP2. The effect of E2F3a on chemotherapeutic sensitivity of ALL cells was implemented by regulating CASP8AP2 expression to a great extent.