CD40 is a costimulatory protein expressed on the surface of many different cells. It delivers signals regulating diverse cellular responses, including proliferation, differentiation, growth suppression, and cell death. In this study, we report a novel CD40 mutant (c.234C>A or p.H78Q) that is expressed in the U266 cell line and in freshly isolated tumor cells. Three-dimensional structural model and Scatchard analysis revealed that the mutated residue located in a region is important for binding to CD40L (CD154). Functional analysis indicated that the mutated CD40 was translocated to the CD40 signalosome and involved in CD40 signal transduction. In conclusion, the mutation in CD40 can lead to an alteration of function, including the change of antigen epitope and the binding affinity with CD40L.