Objective CRISPR/ Cas9 and Cre-loxP were used to construct the conditional knockout of taurine transporter (TauT) in the central nervous system of rats. The mitochondrial mtDNA and mitochondrial respiratory chain enzyme activities in brain tissues were studied. Methods CRISPR/ Cas9 and Cre-loxP techniques were used to obtain heterozygous rats (TauT^{loxP / WT} ) with loxP at both ends of exon 5 of the TauT gene. The obtained TauT^{loxP / WT} rats were mated with Nestin-Cre rats. After breeding and identification, neural-specific TauT gene knockout rats ( TauT^{loxP / loxP / Cre+} ) were obtained. The gene and protein expressions of TauT^{loxP / loxP / Cre+} rats were detected by Real-time PCR, Western blot, and immunohistochemistry. The morphology of brain tissue was observed by hematoxylin-eosin staining. The mtDNA copy number and mitochondrial respiratory chain enzyme ( I/ II/ III/ IV/ V) activity in brain tissue were examined. Results Compared with wild-type rats, TauT gene and protein expressions in TauT^{loxP / loxP / Cre+} rat brain tissues were significantly decreased, and the TauT gene was successfully knocked out in the central nervous system indicating the knockout model was successfully constructed. Hematoxylin-eosin staining showed that the number and density of brain cells were decreased in TauT^{loxP / loxP / Cre+} rats and cytopathic changes more obvious in the brains of old TauT^{loxP / loxP / Cre+} rats. In addition, compared with wild-type rats, the activities of mitochondrial respiratory chain complex enzymes Ⅰ, Ⅲ, Ⅳ, and Ⅴ were significantly decreased, but the copy number of mtDNA was significantly increased. Conclusions The model of TauT gene knockout in the central nervous system of rats was successfully constructed using CRISPR/ Cas9 and Cre-loxP technology. The effects of TauT knockout in the central nervous system on brain tissues, respiratory chain enzymes, and mtDNA of mitochondria were verified, providing a new model platform for the study of the molecular mechanisms of taurine and TauT in brain tissues.