Cancer cells require more nutrients than normal cells to maintain rapid growth and proliferation. L-type amino acid transporter 1 (LAT1: SLC7A5) is highly upregulated in various cancers, and is regarded as a molecular target for cancer therapy. LAT1 preferentially transports large neutral amino acids (Leu, Ile, Val, Met, Phe, Tyr, Trp, and His) including many essential amino acids in a Na⁺-independent manner. JPH203, an LAT1-specific high-affinity inhibitor, strongly suppresses cancer cell proliferation and tumor growth. However, the contribution of LAT1 to the amino acid uptake in cancer cells and the effect of JPH203 on cellular protein synthesis have not been established. Here, we revealed that JPH203 drastically suppresses the uptake of large neutral amino acids into pancreatic cancer cell lines, regardless of the presence or absence of Na⁺. This indicates that LAT1 has the main contribution to cellular amino acid uptake among amino acid transporters including Na⁺-dependent transporters. Furthermore, we revealed, by polysome profiling analysis, that JPH203 treatment suppresses cellular protein synthesis. These results indicate that LAT1 constitutes the main uptake pathway for large neutral amino acids in cancer cells.  Inhibition of LAT1 efficiently suppresses their uptake and reduces the protein synthesis in cancer cells.