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Background and Objectives CD40L is a membrane protein and a member of the tumor necrosis factor super family (TNFSF) that plays an important role in transferring cell signaling in innate and adaptive immunity. Since this protein plays its role through clustering the receptors on the target cell surface, it seems that the multimeric form of this molecule can have a stronger effect than the natural trimeric form. So in this study we describe cloning and expression of dodecameric soluble CD40L through self multimerizing protein, surfactant protein D (SP-D), in the HEK293 cell line. Materials and Methods In this experimental study, the SPD-CD40L was designed in silico and cloned in pcDNA3.1(+) plasmid. HEK293 cells were transfected and used as the expression host cells. The expression of SPD-CD40L was determined at the RNA level by RT-PCR method. After the purification of the recombinant proteins by affinity chromatography, the molecular weight of the recombinant protein was determined by SDS-PAGE. To evaluate the specificity of protein, ELISA and Dot Blot were used. Results The results indicated that HEK293 cells were transfected and recombinant protein was expressed 24 and 48 hours after Transfection. Moreover, the results of ELISA and Dot Blot methods represented the specificity of recombinant SPD-CD40L chimeric protein. Conclusions Chimeric protein SPD-CD40L was expressed successfully by pcDNA3.1(+) in eukaryotic HEK293 cell line and the molecular weight of 4-trimeric (dodecameric) protein was shown to be consistent with the expected amount. [ABSTRACT FROM AUTHOR]