Abstract: Natural rubber is a key strategic material that is vital to the people’s livelihood, the national economy and security. Its primary component is one kind of isoprenoids, known as cis-1,4-polyisoprene. The source of natural rubber is almost exclusive produced from Hevea brasiliensis whose strict growing conditions limit its cultivation to specific tropical environments. The problem of insufficient capacity of natural rubber production become more and more urgent that lead to the search for alternative sources in China. We found that the milk secreted by the laticifers in Periploca sepium contains cis-1,4-polyisoprene, and considered that it may be very suitable for China as a substitute plant for natural rubber production. Two genes encoding PsIPPI and PsCPT, namely isopentenyl diphosphate isomerase and cis-isopentenyl transferase, which play key roles in the biosynthesis of cis-1,4-polyisoprene, were successfully cloned from the branches（including barks, stems and leaves） of P. sepium according to its transcriptome sequencing. Using stem segments as explants, the two genes were overexpressed alone or in combination by Agrobacterium-mediated transformation, and their expression level were increased sharply up to 200-fold. However, the polyisoprene content and the molecular weight distribution had no significant change in the transgenic plants overexpressing PsIPPI alone, while the polyisoprene content and the proportion of high molecular weight increased about 5-fold and 1.6-fold in those of transgenic plants overexpressing PsCPT alone, as well as increased about 5-fold to 6-fold and 2-fold in those of PsIPPI and PsCPT co-overexpressing transgenic plantlets. In current research, the functions of the key genes associated with the biosynthesis of cis-1,4-polyisoprene in P. sepium were preliminarily identified, and the possibility was proved that the yield and high molecular weight of natural rubber could be improved by overexpression of some key genes. With the progress in the research on cis-1,4-polyisoprene biosynthesis mechanism, more and more importent enzymes and their encoded genes will be discovered and characterized, it is expected that P. sepium will be developed as an alternative source for the production of natural rubber, and completely solve the bottleneck of natural rubber existing in China.