Epstein-Barr virus (EBV)-based vectors can stably maintain large genomic fragments in mammalian cells, offering great potential for the treatment/correction of many acquired and inherited disorders. Numerous studies report marked increases in the transfection efficiency of EBV-based vectors after delivery into cell lines constitutively expressing Epstein-Barr nuclear antigen-1 (EBNA1), compared with cells not expressing EBNA1. We employ a novel strategy, involving the transfection of mRNA encoding EBNA1, to transiently express EBNA1 protein in human cells. Subsequently we show that the transfection efficiency of a 21-kb EBV-based vector is improved significantly when codelivered with mRNA encoding EBNA1. Similar increases in transfection efficiency were observed after delivery of the plasmid into cells constitutively expressing EBNA1. We also investigate the mechanism by which EBNA1 facilitates the transfection of EBV-based vectors, using mRNA encoding modified versions of the protein. Previous studies suggest that the EBNA1 DNA-binding domain (DBD), together with the nuclear localization signal (NLS), may enhance transfection of EBV plasmids by facilitating their nuclear transport. We demonstrate that an EBNA1 derivative comprising only the NLS and DBD does not facilitate transfection of EBV-based vectors. However, cells expressing an EBNA1 derivative devoid of a functional NLS but retaining the chromatin-binding regions, domains A and B, enhances plasmid transfection efficiency by up to 10-fold. Moreover, a variant of EBNA1 comprising two copies of domain A fused to the DBD enhances DNA transfection to an even greater extent than wild-type EBNA1. We therefore propose that EBNA1-mediated transfection of EBV-based vectors is dependent on the presence of chromatin-binding regions and the DBD, but not the NLS.