Abstract: CD154 plays an important role in the interaction between antigen-specific T lymphocytes and antigen-presenting cells. In this study, the full-length cDNA encoding chicken CD154 was amplified from isolated peripheral blood mononuclear cells (PBMC) of Sanhuang chicken using RT-PCR- Sequence analysis showed that the chicken CD154 is comprised of 819 base pairs in length and encodes a 272 amino acid (aa) protein.The full-length cDNA fragment of chicken CD154 was further subcloned into prokaryotic expression vector pET-28a, resulting in the expression plasmid pET-28a-CD154. However, no specific expressed protein was detected after transformation of BL21(DE3). The truncated fragment of chicken CD154 (CD154d) without signal peptide (1-22 aa) and transmembrance region (23-46 aa) was cloned using PCR and further subcloned into downstream of hexahistidine sequence of pQE80. Expression of a fusion protein (6×His-CD154d) with 27 ku molecular mass was observed in E. coli DH5α, and the result of Westernblot demonstrated that the fusion protein can be recognized by anti-6×His monoclonal antibody. In addition, the fulllength cDNA fragment of chicken CD154 was inserted into downstream of EGFP gene of eurokaryotic expression vector pEGFP-C1, resulting in the expression plasmid pEGFP-C1-CD154. After transfection in Hela cells, the fluorescence of the fusion protein EGFP-CD154 was observed on cell membrane by fluorescent microscope, indicating that the chicken CD154 is a membrane-associated protein. The present study laid a foundation for further investigating the structure and function of chicken CD154 and its adjuvant potency in chicken vaccines.