The bursa of Fabricius (BF) is a central immune organ in birds, and some peptides from chicken BF have demonstrated important immune functions. Here, a new 626.27 Da pentapeptide, Bursopentin (BP5, Cys-Lys-Arg-Val-Tyr) was isolated and purified by reverse-phase high-performance liquid chromatography. In this study, we examined the effects of BP5 on antigen-specific immune response in BALB/c mice sensitized with inactivated avian influenza virus (AIV) [A/Duck/Jiangsu/NJ08/05 (AIV H9N2 subtype)]. The results suggested that BP5 enhanced anti-hemagglutinin antibody (IgG, the isotypes IgG1 and IgG2a) production, induced both of Th1- (IL-2 and IFN-γ) and Th2-type (IL-4 and -10) cytokines, increased proliferations of splenic lymphocyte subsets CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+) and B cells, and enhanced cytotoxic T-lymphocyte activity of the activated splenocytes against NIH3T3 cells. The effects of BP5 on the proliferation of isolated T- and/or B-cell populations of BALB/c mice were assessed, and the data suggested that BP5 promoted spleen lymphocyte proliferation by activating B cells directly and T cells indirectly. Further analysis revealed that B-lymphocyte proliferation induced by BP5 is mediated by reactive oxygen species generated from thiol auto-oxidation of BP5. Furthermore, our data indicated that protein kinase C, mitogen-activated protein kinase, and nuclear factor kappa B are involved in the signal transductions during the BP5-induced B lymphocyte proliferation. This study indicates that BP5 could be a potential immunomodulator for future immuno-pharmacological use.