(1) Background and aim: Aloe arborescens Mill. ( A. arborescens ) is one of the most widely distributed species in the genus Aloe and has garnered widespread recognition for its anticancer properties. However, the molecular mechanisms underlying these activities have not yet been fully elucidated. This study aimed to explore the effects of the plant polar glycosidic fraction (AAG) on hepatocellular carcinoma (HCC) in an in vivo model induced by diethylnitrosamine (DEN). (2) Experimental procedure: The fraction was standardized using HPLC-PDA-MS/MS fingerprinting, and two distinct intragastric AAG dose regimens were examined (10 and 20 mg/kg) in combination with DEN 200 mg/kg. Serum alpha-fetoprotein (AFP), gamma-glutamyl transferase (γ-GGT), glutathione S-transferase placental (GST-P), mRNA expression of metabolic cytochrome enzymes (CYP1A3 and CYP2B2), inflammatory genes (nuclear factor kappa-B p65 subunit
NF-κB p65), metalloproteases 9 (MMP9), tissue inhibitors of metalloproteases (TIMP1), transforming growth factor beta 1 (TGFβ1), and histological features were assessed. (3) Key results and conclusions and implications: AAG was characterized by five major secondary metabolites: saponins, chromones, anthraquinone, and triterpenes. The fraction reduced hepatic malignancy characteristics by diminishing the size and number of altered foci and lowering hepatic cancer biomarkers, such as γ-GGT, AFP, and GST-positive foci. It also reduced the mRNA levels of CYP1A3 and CYP2B2, NF-κB p65, and MMP9, hepatic Ki-67, and TGFβ1 while upregulating TIMP1 levels. This study revealed that AAG exhibited a marked suppressive effect on HCC cell proliferation, displaying a range of mechanistic actions, including decreasing the metabolic activation of cytochrome enzymes, which consequently reduced the production of reactive oxygen species and other genes implicated in cancer development. AAG could be a significant therapeutic candidate for patients diagnosed with hepatocarcinoma.