Raltitrexed (RTX) is an antimetabolite drug used as a chemotherapeutic agent for treating colorectal cancer, malignant mesothelioma, and gastric cancer. The antitumor capacity of RTX is attributed to its inhibitory activity on thymidylate synthase (TS), a key enzyme in the synthesis of DNA precursors. The current study is aimed at investigating the potential antitumor effects of RTX in liver cancer. Using the HepG2 cell line as an in vitro model of liver cancer, we evaluated the effects of RTX on cell proliferation employing both a WST-8 assay and a clone formation efficiency assay. In addition, we monitored the ultrastructure changes of HepG2 cells in response to RTX with transmission electric microscopy. To investigate the mechanism underlying the regulation of cell proliferation by RTX, we analyzed cell cycle using cell flow cytometry. Moreover, real-time PCR and Western blot analyses were conducted to examine expression levels of cell cycle regulatory proteins cyclin A and cyclin-dependent kinase 2 (CDK2), as well as their mediators tumor suppressor genes p53 and p16. Our results demonstrate that RTX inhibits HepG2 proliferation by arresting the cell cycle at G0/G1. This cell cycle arrest function was mediated via downregulation of cyclin A and CDK2. The observed elevated expression of p53 and p16 by RTX may contribute to the reduction of cyclin A/CDK2. Our study indicates that RTX could serve as a potential chemotherapeutic agent in the treatment of hepatocellular carcinoma.