This chapter summarizes the study on related plasmids of Bifidobacterium expression plasmids in Bifidobacterium for cancer gene therapy, and the advantages and disadvantages of the use of Bifidobacterium as a delivery system of functional genes for cancer gene therapy. To develop a cloning vector of Bifidobacterium, a comprehensive understanding of the replication mechanism and characterization of natural bifidobacterial plasmids is necessary. Bifidobacterium is gram-positive, a strictly anaerobic bacterium with bifid morphology. As an anaerobe, Bifidobacteria can germinate and proliferate in the hypoxic regions of solid tumors. Plasmids of Bifidobacterium encode many specific characteristics of Bifidobacterium, and they can also establish shuttle vectors to express exogenous genes. As a delivery system and compared with other bacterial genera, Bifidobacterium represents a promising biological tool for cancer therapy. Bifidobacterium with functional genes can reduce the side effects and is propitious to cancer therapy. The combination of Bifidobacterium with conventional chemotherapy drugs and radiation is a potential tool to destroy tumor cells more drastically.