Among viral and non-viral gene delivery systems, SV40-based vectors show great promise in the cancer gene therapy field. SV40 vectors very efficiently deliver genes such as anti-viral agents, DNA vaccine, genes for chemoprotection (such as ABC transporters genes), suicide genes and antiangiogenic genes. The recombinant SV40 vectors can infect a wide variety of cells-dividing cells as well as non-cycling ones. Most of the SV40-based vectors can incorporate larger transgenes than the capacity of the SV40 wild-type, which is 5.2 kb; Moreover, in vitro packaged vectors demonstrate efficient delivery of plasmids with a molecular weight of up to 17.7 kb. SV40-based vectors carry some SV40 viral sequences, but the SV40 in vitro-packaged vectors are free of any SV40 wild-type viral DNA sequences. These vectors are prepared with nuclear extracts of SF9 insect cells containing the main viral capsid protein of the SV40 wild-type virus, VP1. This review summarizes different strategies in which SV40 vectors are used to deliver genes in vitro, to living mice, and to tumors growing in nude mice.