Hepatocellular Carcinoma (HCC) is extremely aggressive and presents low rates of response to the available chemotherapeutic agents. Many studies have focused on the search for alternative low-cost natural compounds with antiproliferative potential that selectively respond to liver cancer cells. This study assessed the in vitro direct action of trans-chalcone (TC) on cells of the human HCC HuH7.5 cell line. As methods, we subjected the HuH7.5 tumor cells to TC treatment at increasing concentrations (12.5–100 µM) for 24 and 48 h. Cell viability was verified through MTT and 50% inhibitory concentration of cells (IC50 23.66 µM) was determined within 48 h. We quantified trypan blue proliferation and light microscopy, ROS production, mitochondrial depolarization and autophagy, cell cycle analysis, and apoptosis using Muse® cell analyzer and immunocytochemical markings of p53 and β-catenin. In the results, data showed an effective dose- and time-dependent TC-cytotoxic action at low micromolar concentrations without causing toxicity to non-cancerous cells, such as erythrocytes. TC-treatment caused mitochondrial membrane damage and cell cycle G0/G1 phase arrest, increasing the presence of the p53 protein and decreasing β-catenin, in addition, to inducing cell death by autophagy. Additionally, TC decreased the metastatic capacity of HuH7.5, which affected the migration/invasion of this type of cell. We had as conclusion that: In vitro TC activity in the human HCC HuH7.5 tumor cell line is shown to be a potential molecule to develop new therapies to repair the p53 pathway and prevent the overexpression of Wnt/β-catenin tumor development inducing autophagy cell death and decreasing metastatic capacity of HuH7.5 cell line.