Background/Objectives: Cholesterol is vital in various bodily functions, such as maintaining cell membranes, producing hormones, etc. However, imbalances, like hypercholesterolemia, can lead to diseases such as cancer, kidney disease, non-alcoholic fatty liver disease, and cardiovascular conditions. This study explores the impact of kiwifruit consumption, specifically Actinidia arguta cultivar Geneva and Actinidia deliciosa cultivar Hayward, on cholesterol and lipid metabolism in rat liver. Methods: Rats were divided into groups: a 1% cholesterol control group (Ch), a 5% Geneva kiwifruit-supplemented group (ChGENE), and a 5% Hayward kiwifruit-supplemented group (ChHAYW). Gene expression was analyzed using Gene Spring v.14. Gene ontology, pathway analysis, miRNA, and transcription factor prediction were performed using DAVID, Reactome, and miRNet. In addition, we used Agilent Literature Search software to gain further insights. Results: Statistical analysis identified 72 genes in ChGENE-Ch and 2 genes in ChHAYW-Ch comparison. Key genes involved in cholesterol metabolism pathways, including PCSK9, SCD1, SLC27A5, HMGCR, and DHCR24, showed lower expression in the kiwifruit-supplemented groups. The genes mentioned above showed lower expression in the kiwifruit-supplemented group, probably contributing to the liver lipid level reduction. Further analysis identified miRNA-26a, miRNA-29a/b/c, miRNA-33a/b, and miRNA-155 targeting hub genes. Conclusions: Our findings suggest that dietary supplementation with kiwifruit, particularly the Geneva cultivar, reduces fat accumulation in the liver of rats with hypercholesterolemia, likely through downregulation of critical genes involved in cholesterol metabolism. These studies highlight the potential of kiwifruit as a part of a dietary strategy to manage cholesterol levels.