Recent research has shown that the Na⁺/K⁺-ATPase α1 subunit is a novel anti-cancer target, which plays pivotal roles in malignant cell ion transport, metabolism, migration and signal transduction. The purpose of the present study was to investigate the anti-cancer effects of ouabain and Na⁺/K⁺-ATPase α1 small interfering ribonucleic acid (siRNA) on HepG2 cell proliferation, apoptosis and cell cycle, and to explore the molecular mechanisms. The expression of Na⁺/K⁺-ATPase α1 subunit in human hepatocellular carcinoma (HCC), normal liver tissues and human HCC line (HepG2, SMMC-7721 and Bel-7402) has been investigated. Using the ouabain and Na⁺/K⁺-ATPase α1 subunit siRNA, which target the Na⁺/K⁺-ATPase, we have evaluated the effects of inhibiting Na⁺/K⁺-ATPase α1 in human HepG2 cells with respect to cell proliferation, morphology, cell cycle, impact on intracellular Ca2++, reactive oxygen species (ROS) concentration, and correlated gene expression level on messenger ribonucleic acid (mRNA) and protein. Our data showed that the expression Na⁺/K⁺-ATPase α1 subunit in HCC tissues is higher than that in normal liver tissues. Ouabain and Na⁺/K⁺-ATPase α1 siRNA could inhibit HepG2 cell proliferation. Ouabain could induce HepG2 cell apoptosis and generate S phase arrest, and siRNA could enhance the anti-cancer effect of ouabain that induced HepG2 cells apoptosis via an intracellular Ca²⁺ and ROS increase-mediated, and generated cell cycle S phase arresting by decreasing the CyclinA1/cyclin-dependent kinase 2 (CDK2)/proliferating cell nuclear antigen (PCNA) complex product and increasing the expression of cyclin-dependent kinase inhibitor 1A (P21^CIP1). We believe that targeting of the Na⁺/K⁺-ATPase α1 subunit in human HCC cells could provide new sight into the treatment of HCC.