Hepatocellular carcinoma (HCC) is one of the most common cancer types, and chemotherapy plays an important role in treatment of HCC. However, long‑term treatment with chemotherapeutic drugs such as 5‑fluorouracil (5‑FU) often results in chemoresistance, and the underlying mechanisms remain unclear. In this study, we showed that the annexin A2 (ANXA2) protein is highly expressed in hepatoma cells compared to healthy cells. Knockdown of the ANXA2 gene inhibited hepatoma cell growth, and the underlying mechanism may involve cell cycle inhibition through downregulation of β‑catenin and cyclin D1. We also investigated the role of ANXA2 in chemotherapeutic treatment with 5‑FU. 5‑FU inhibited hepatoma cell growth, while ANXA2 overexpression reduced, and knockdown enhanced, the effects of 5‑FU on hepatoma cell growth. Furthermore, β‑catenin and cyclin D1 were asscociated with the ANXA2‑induced resistance. Taken together, our data suggest that the ANXA2 protein is a critical factor in HCC and that its downregulation can enhance chemotherapeutic treatment with 5‑FU. ANXA2 may thus constitute a new therapeutic target for HCC.