〔Abstract〕 Objective To construct a knockdown recombinant adeno-associated virus(AAV-shCTSK) targeting the mouse Cathepsin K(CTSK)gene and assess the knockdown efficiency of AAV-shCTSK in mice, and to investigate its impact on lipid storage within adipose tissue. Methods ShRNA primers specific to both the negative control(NC) and CTSK were designed, annealed, and integrated into the backbone vector. Following clone selection and sequencing for validation, recombinant plasmids were purified. Adeno-associated viral vectors, along with packaging and helper plasmids, were co-transfected into 293T cells using the transfection reagent PEI for adeno-associated viral packaging and amplification.The resultant AAV-shNC and AAV-shCTSK were injected into the epididymal adipose tissue of miceviain situ injection. Two weeks post-injection, the expression of CTSK protein was evaluated through immunoblotting assay and the size of intracellular lipid droplets in mouse adipose tissue was detected by HE staining. Results Successful acquisition of AAV-shNC and AAV-shCTSK adeno-associated viruses was achieved. In mice subjected to in situ injection of AAV-shCTSK, effective knockdown of CTSK in adipose tissue was confirmed, accompanied by a significant reduction in the size of white adipocytes. Conclusion A knockdown adeno-associated virus targeting CTSK in mice is successfully constructed, and CTSK knockdown in adipose tissue leads to a notable decrease in lipid content.