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In this study, we established a novel reproducible method for<jats:named-content content-type=\"genus-species\">C. pneumoniae<\/jats:named-content>transformation based on a plasmid shuttle vector system. We constructed a<jats:named-content content-type=\"genus-species\">C. pneumoniae<\/jats:named-content>plasmid backbone shuttle vector, pRSGFPCAT-Cpn. The construct expresses the red-shifted green fluorescent protein (RSGFP) fused to chloramphenicol acetyltransferase in<jats:named-content content-type=\"genus-species\">C. pneumoniae<\/jats:named-content>.<jats:named-content content-type=\"genus-species\">C. pneumoniae<\/jats:named-content>transformants stably retained pRSGFPCAT-Cpn and expressed RSGFP in epithelial cells, even in the absence of chloramphenicol. The successful transformation in<jats:named-content content-type=\"genus-species\">C. pneumoniae<\/jats:named-content>using pRSGFPCAT-Cpn will advance the field of chlamydial genetics and is a promising new approach to investigate gene functions in<jats:named-content content-type=\"genus-species\">C. pneumoniae<\/jats:named-content>biology. 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