The absence of tools for the genetic manipulation of C. pneumoniae has hampered research into all aspects of its biology. In this study, we established a novel reproducible method for C. pneumoniae transformation based on a plasmid shuttle vector system. We constructed a C. pneumoniae plasmid backbone shuttle vector, pRSGFPCAT-Cpn. The construct expresses the red-shifted green fluorescent protein (RSGFP) fused to chloramphenicol acetyltransferase in C. pneumoniae . C. pneumoniae transformants stably retained pRSGFPCAT-Cpn and expressed RSGFP in epithelial cells, even in the absence of chloramphenicol. The successful transformation in C. pneumoniae using pRSGFPCAT-Cpn will advance the field of chlamydial genetics and is a promising new approach to investigate gene functions in C. pneumoniae biology. In addition, we demonstrated that pRSGFPCAT-Cpn overcame the plasmid species barrier without the need for recombination with an endogenous plasmid, indicating the potential probability of horizontal chlamydial pathogenic gene transfer by plasmids between chlamydial species.