Abstract: Objective To investigate the effects of Cytosine arabinoside (Ara-C) on co-stimulating molecule CD80 and CD86 in human B leukemia cell line Nalm-6 and the cytotoxicity of T lymphocytes mediated by bispecific antibody combined with Ara-C on Nalm6 cells in vitro. Methods Inhibitory growth of Nalm-6 induced by Ara-C was measured by MTT. Change of CD80 and CD86 expression in Nalm-6 stimulated by Ara-C were detected by FACS. The cytotoxicity of T lymphocytes mediated by bispecific antibody combined with or without Ara-C on lysing the target cells was determined by non-radioactive fluorescence method. The changes of interleukin 2 (IL-2) secreted or CD25 and CD69 expression in activated T cells were measured by FACS and ELISA. Results The IC10 of Ara-C on Nalm-6 cells growth was 0.25 μM. Ara-C could upregulate CD80 expression in leukemia cell line Nalm-6. The cytotoxicity rate of T cells mediated by bispecific antibody was enhanced along with the increase of E/T ratio. The combination of antiCD3×antiCD19 bispecific antibody and Ara-C showed the greatest effectiveness in enhancing the cytotoxicity of T cells against the tumor target cells in vitro. Activated T cells expressed higher levels of CD25 and CD69 and released more IL-2. Conclusion Ara-C could up-regulate CD80 expression in CD19+ Nalm-6 cells, thus enhanced the cytotoxicity of T lymphocytes mediated by bispecific antibody and activated T cells more effectively.