Simple Summary: Liver fibrosis is a chronic disease that can lead to serious health problems, including liver cancer. However, the pathogenesis of liver fibrosis caused by natural aging is not fully understood. For researchers studying liver fibrosis, an important tool for conducting mechanism research and testing anti-liver fibrosis therapies is an in vivo model that can reflect the pathological causes of liver fibrosis. This study aimed to establish a model of spontaneous liver fibrosis in NOD/SCID mice induced by natural aging. This model's validity was confirmed using cutting-edge fibrosis-related measurements. This study provides a useful tool for further research into the causes and potential treatments for liver fibrosis. The findings may help to develop new therapies for chronic liver diseases which affect millions of people worldwide. Liver fibrosis, a critical pathological feature of chronic liver diseases, arises from a multitude of pathogenic factors. Consequently, establishing an appropriate animal model to simulate liver fibrosis holds immense significance for comprehending its underlying pathogenesis. Despite the numerous methodologies available for generating liver fibrosis models, they often deviate substantially from the spontaneous age-related liver fibrosis process. In this study, compared with young (12 weeks) and middle-aged NOD/SCID mice (32 weeks), there were a large number of fibrous septum and collagen in the liver tissue of old NOD/SCID mice (43 weeks, 43 W). Immunohistochemical analysis unequivocally indicated heightened α-SMA content within the liver tissue of the 43 W mice, thereby underscoring aging's role in triggering the epithelial-to-mesenchymal transition. In addition, SA-β-gal staining as well as P21 expression were increased, and SIRT1 and SIRT3 expression were decreased in 43 W mice. A comprehensive evaluation encompassing transmission electron microscopy and fluorescence quantitative analysis elucidated compromised mitochondrial function and reduced antioxidant capacity in hepatocytes of the 43 W mice. Furthermore, the aging process activated the pro-fibrotic TGF-β-SMAD pathway, concurrently inducing hepatocellular inflammation. The results of the present study not only validate the successful construction of a spontaneous liver fibrosis mouse model through natural aging induction but also provide initial insights into the mechanisms underpinning age-induced liver fibrosis. [ABSTRACT FROM AUTHOR]