Background/Objectives: Increasing incidences of hepatocellular carcinoma (HCC) and its complicated treatment protocols promote novel drug discovery programs. Maslinic acid (MA) is a naturally occurring oleane-type triterpenoid derived mainly from Olea europaea L., with evident antineoplastic potential. Aims: This study aimed to investigate in vitro, in silico, and in vivo antineoplastic activity of MA from olive leaves. Methods: The in vitro antiproliferative activity of MA on HCC cell line (HepG-2) was investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The study comprised an in silico exploration of the putative targets of MA predicted via open-access databases in order to reach a refined list of genes/proteins that would be later investigated to explore the predisposed pathways guiding the experiment forward. The novel antineoplastic molecular mechanisms of MA were further evaluated in vivo against diethylnitrosamine (DEN)-induced HCC probably mediated by targeting hepatic ATP citrate lyase (ACLY)/Wnt/β-catenin pathway for the first time in rats, when administered on the 12th week of the experiment model (50 mg or 100 mg/kg/day orally). Results: MA showed significant anticancer activity against HepG-2 cancer cell line with the concentration required for 50% growth inhibition (IC50) value of 18.6 μg/mL, compared to the reference drug doxorubicin, which had an IC50 value of 3.181 μg/mL. The in silico prediction results illustrated that most of the acknowledged genes/proteins were implicated and enriched in cancer pathways, regulation of inflammatory response and cellular response to stress. Wnt–catenin pathway and apoptosis-related markers were furthermore investigated experimentally. MA downregulated ACLY expression, switching off both Wnt arms and stabilizing cell death machinery. Furthermore, MA modulated hepatocellular oxidative and inflammatory responses. Additionally, this overall state was reflected positively, displaying conservation of the liver histopathological architecture. Conclusions: The study showed new evidence for the potential of MA to ameliorate DEN-induced HCC; therefore, MA is a promising antitumor agent for attenuating HCC.