Interleukin 15 (IL-15) is a pivotal cytokine for the proliferation and activation of a specific group of immune cells such as natural killer (NK), IFN-producing killer dendritic cells (IKDC) and CD8 T cells. RAE-1epsilon, the ligand for the activating NKG2D receptor, which also play an important role in the proliferation and activation of NK cells and IKDCs. In this study, a membrane-bound form of IL-15 (termed mb15) encoding sequence and RAE-1epsilon gene were obtained by SOE-PCR or PCR amplification. The amplified mb15 and RAE-1epsilon gene were then digested and inserted into the multiple cloning site1 (MCS1) and MCS2 of pVITRO2-mcs vector, respectively. A recombinant eukaryotic expression vector for co-expression of mb15 and RAE-1epsilon was successfully constructed. After it was transfected to BaF3 cells, the expression of IL-15 and RAE-1epsilon in recombinant BaF3/mb15/RAE-1epsilon cells were verified by RT-PCR, western blot and FCM analysis. Furthermore, BaF3/mb15/RAE-1epsilon cells had the ability of promoting NK cells proliferation and IFN-gamma secretion. In conclusion, BaF3/mb15/RAE-1epsilon cells were successfully constructed, which is very useful for further studies, especially for the expansion and activation of certain subsets of immune cells such as NK cells and IKDCs.