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Abstract Cardiac fibroblasts (CFs) are an attractive target for reducing pathological cardiac remodeling, and understanding the underlying mechanisms of these processes is the key to develop successful therapies for treating the pressure‐overloaded heart. CF‐specific knockout (KO) mouse lines with a Cre recombinase under the control of human TCF21 (hTCF21) promoter and/or an adeno‐associated virus serotype 9 (AAV9)‐hTCF21 system provide a powerful tool for understanding CF biology in vivo. Although a variety of rat disease models are vital for the research of cardiac fibrosis similar to mouse models, there are few rat models that employ cardiac cell‐specific conditional gene modification, which has hindered the development and translational relevance of cardiac disease models. In addition, to date, there are no reports of gene manipulation specifically in rat CFs in vivo. Here, we report a simplified CF‐specific rat transgenic model using an AAV9‐hTCF21 system that achieved a CF‐specific expression of transgene in adult rat hearts. Moreover, we successfully applied this approach to specifically manipulate mitochondrial morphology in quiescent CFs. In summary, this model will allow us to develop fast and simple rat CF‐specific transgenic models for studying cardiovascular diseases in vivo.