CpG oligonucleotide (CpG-ODN) can exert an immunostimulatory effect on different types of immune cells such as dendritic cells (DC). The immunostimulatory activity of CpG-ODN is closely related to its nucleotide sequence and structural characteristics. In this study, we aimed at evaluating the stimulatory effects of different CpG-ODN on the maturation of chicken bone marrow-derived DC (BM-DC) in vitro. First, 4 CpG-ODN were designed. Then chicken bone marrow cells were extracted from tibia and femur and cultured in the RPMI 1640 medium with recombinant chicken granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4. After culture for 6 d, the cells were stimulated by different CpG-ODN or lipopolysaccharide for 24 h. Finally, the effects of different CpG-ODN on the maturation of chicken BM-DC were investigated by morphologic, phenotypic, and functional assays. The results showed that the cultured cells could display the typical DC morphology, and the CpG-ODN could efficiently stimulate the BM-DC to show the mature morphologic characteristics and upregulate the expression of cluster of differentiation (CD) 40 and CD86 molecules. In addition, after stimulation by CpG-ODN, the BM-DC could significantly induce T-cell proliferative response (P < 0.01). Among all the sequences, the stimulatory effect of CpG-ODN F3 with an addition of poly-guanosine strings at the 3' end was the best on the chicken BM-DC. In conclusion, this is the first report to demonstrate that different CpG-ODN have distinct stimulatory effects on the maturation of chicken BM-DC and CpG-ODN F3 with the best stimulatory effect can be a potent stimulant for the maturation of chicken BM-DC.