Abstract: Background　There is currently a lack of animal models of primary liver cancer that can recapitulate human metabolic characteristics. Guinea pigs, which exhibit high similarity to humans in terms of metabolism and immunity, represent an urgent need in translational medicine to establish a mature hepatocellular carcinoma (HCC) model. Objective　This study aimed to establish a guinea pig PLC model using diethylnitrosamine (DEN) induction (DEN) induction. Methods　Forty-eight 2-week-old male guinea pigs were randomly divided into four groups (n = 12): Group Ⅰ (control group) received intraperitoneal (i.p.) injection of normal saline; Group Ⅱ received i.p. injection of 50 mg/kg DEN (twice a week for 4 weeks) with normal drinking water; Group Ⅲ received the same injection regimen as Group Ⅱ but was given alternating normal drinking water and drinking water containing 100 μg/L DEN daily; Group Ⅳ received i.p. injection of 100 mg/kg DEN (twice a week for 4 weeks) with normal drinking water. At six months post-modeling, survival rates were calculated, blood samples were collected after euthanasia, and liver tissues were harvested for gross morphology observation and tumor incidence. Hematoxylin and eosin (HE) staining was performed, and liver function and inflammatory factors were measured by ELISA. Immunohistochemistry was used to detect Ki-67 and proliferating cell nuclear antigen (PCNA) expression. Results　Survival rates in Groups I and Ⅱ were 100%, and no tumors were observed. Mortality rates in Groups Ⅲ and Ⅳ were 16.67% and 58.33%, respectively, with tumor incidence rates of 90% and 60% (P＜0.05). Compared with Group Ⅰ, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were elevated in Groups Ⅱ, Ⅲ, and Ⅳ (P＜0.05). Levels of tumor necrosis factor-alpha (TNF- α), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and C-reactive protein (CRP) were increased in Groups Ⅲ and Ⅳ (P＜0.05). Ki-67 and PCNA-positive cells were also increased. Conclusion　Low-dose DEN injection combined with drinking water exposure (Group Ⅲ) can successfully establish a guinea pig model of liver cancer, achieving both high tumorigenicity and low mortality, thereby providing a novel modeling strategy for translational medicine.