Abstract： AIM: To amplify and express human CD40L gene in the plasma membrane of NIH3T3 for an easy and effective method to produce human CD40L protein. METHODS: Human CD40L gene was amplified by gradient RT-PCR from Jurkat cell line and subcloned into the T vector. After sequencing, pcDNA3. 1-CD40L was constructed and transfected into NIH3T3 cell line. The expression of CD40L was tested by FACS. RESULTS: The CD40L gene was successfully amplified from Jurkat cell line. Sequencing identified a clone with CD40L gene without any muta-tion. After transfection into NIH3T3 and primary selection with G418, about 41% cells successfully transported the expressed CD40L protein to the plasma membrane. CONCLUSION: Human CD40L gene can be amplified from Jurkat cell line. pcDNA3.1 vector expresses CD40L successfully in NIH3T3 cell line and the expressed protein is partially transported to plasma membrane.