Gene transfer of CD40 ligand (CD40L) holds promise as a novel therapy for lymphoid malignancies and a number of solid carcinomas because of its multiple anti-tumor activities. However, membrane-bound CD40L can be cleaved into a soluble form, sCD40L, which contributes to systemic inflammatory and cardiovascular diseases, and induces survival signals in the absence of protein synthesis block, suggesting a deleterious side effect of CD40L gene therapy. We generated a plasmid encoding non-cleavable human CD40L mutant (pcDNA3.1+-CD40L-M) to determine the direct anti-proliferative and pro-apoptotic effects in CD40-positive lung adenocarcinoma cell line A549, to verify activation of immature dentritic cells (DCs) by co-cultivation with the transfected A549 cells and to evaluate the lower expression of sCD40L relative to that of wild-type CD40L (CD40L-WT) transfectant in cell-free supernatants. These studies suggest that gene transfer of the membrane-stable CD40L mutant into CD40-positive cells may provide an efficient and safe method to treat non-small cell lung cancer.