Hepatocellular carcinoma (HCC) is the most frequent type of liver cancer and accounts for 90% of all liver cancers. Although strategies and outcomes for patients afflicted with HCC have improved recently, long-term prognoses remain bad, in part because of the lack of effective approaches for early diagnosis. Therefore, the development of new drugs is expected. Choline is one of the biofactors that plays an important role in the structure and the function of biological membranes, and it is essential for the synthesis of phospholipids. Increased choline accumulation has been identified in various tumors. However, the molecular mechanisms of choline transporters in HCC have not been elucidated. We examined the molecular and functional analyses of choline transporters in human HCC cell line HuH-7 and the elucidation of the action mechanism behind the antitumor effect of novel choline-transporter-like protein 1 (CTL1) inhibitors, Amb4269951 and Amb4269675. CTL1 was highly expressed in HuH-7 cells and localized to the plasma membrane. Choline uptake was characterized by Na⁺-independence, a single-uptake mechanism. Choline deficiency inhibited cell viability and increased caspase-3/7 activity, suggesting that the inhibition of CTL1 function, which is responsible for choline transport, leads to apoptosis-induced cell death. Both Amb4269951 and Amb4269675 inhibited choline uptake and cell viability and increased caspase-3/7 activity. CTL1 is a target molecule for treating HCC, and its inhibitors Amb4269951 and Amb4269675 are novel lead compounds.