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Two-dimensional shear wave elastography (2-D SWE) has been proven to detect hyperlipidemia-induced elastic abnormality in the corpus cavernosum. This study investigated cytological factors affecting the elasticity of the corpus cavernosum in rabbits with hyperlipidemia using single-cell RNA sequencing (scRNA-seq). Male New Zealand white rabbits were randomly divided into a hyperlipidemia group (high-cholesterol diet) and a control group (standard diet). Penile 2-D SWE was performed to detect the elastic abnormality in the corpus cavernosum. ScRNA-seq was performed to observe cellular changes in the corpus cavernosum of rabbits with hyperlipidemia. Immunohistochemistry, immunofluorescence and histological examinations were conducted to verify the results of scRNA-seq. Two-dimensional SWE revealed that the Young's modulus of the corpus cavernosum was significantly greater in the hyperlipidemia group than that in the control group (p < 0.001). Histological findings revealed extracellular matrix accumulation within the corpus cavernosum, with stronger staining of collagen types I and Ⅲ. ScRNA-seq revealed that fibroblasts, smooth muscle cells, and endothelial cells were the major cell types in the corpus cavernosum. A novel subtype of fibroblasts (myofibroblast) was discovered in the hyperlipidemia group, which was verified by immunofluorescence staining and gene ontology analysis. Fibroblasts, smooth muscle cells and endothelial cells were three cellular sources for myofibroblasts. Myofibroblasts are activated and proliferate and secrete large amounts of collagen fibers in the corpus cavernosum during hyperlipidemia, leading to abnormal Young's modulus detected by 2-D SWE and their recognition as a new factor affecting the hyperlipidemia-induced elastic abnormality of the corpus cavernosum. [ABSTRACT FROM AUTHOR]