Abstract Background: The KRAS gene is mutated in approximately 40% of colorectal cancer (CRC). Patients with KRAS mutant CRC do not benefit from treatment with anti-EGFR antibody cetuximab and panitumumab. TAS-102 is a novel oral anti-neoplastic nucleoside agent, consisting of trifluorothymidine (FTD) as the antitumor component and thymidine phosphorylase inhibitor which prevents degradation of FTD. In a previous report, TAS-102 showed a broad spectrum of antitumor effect against various types of tumors via FTD incorporation into DNA followed by possible DNA dysfunction. Here, we investigated TAS-102 antitumor potency against colorectal cancer cell lines with differing KRAS status. Materials and Methods: Cytotoxicity of FTD and cetuximab by 72h treatment was evaluated using cancer cell lines with varying of KRAS gene status. The antitumor effect of TAS-102 and cetuximab was compared in mice subcutaneously implanted with colorectal cancer cell lines with KRAS wild-type or mutation. In the same models, the amount of FTD incorporated into DNA was analyzed by HPLC in the TAS-102-treated tumors. EGFR downstream signaling pathways were analyzed by immunoblotting in the cetuximab-treated tumors. Results: KRAS wild-type cell lines were sensitive to cetuximab in vitro, while some of the KRAS mutants were not. Cetuximab showed antitumor effect against KRAS wild-type tumors with downregulation of EGFR downstream signaling pathways, and it didn't show antitumor effect against KRAS mutants. In contrast to cetuximab, FTD exhibited a broad spectrum of cytotoxicity in vitro irrespective of KRAS status. Additionally, TAS-102 exerted significant antitumor effect against KRAS wild-type and mutant tumors. FTD incorporation into DNA was observed in both tumor types. Conclusion: TAS-102 appears to exert significant antitumor effects on CRC cell lines irrespective of KRAS status which is consistent with preliminary clinical findings. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B86. Citation Format: Nozomu Tanaka, Yukari Yamada, Akio Fujioka, Keisuke Yamamura, Satoko Ito, Kenichi Matsuo, Teruhiro Utsugi. Potent antitumor effect of TAS-102, a novel oral anti-neoplastic nucleoside agent, on both cetuximab-sensitive KRAS wild-type and cetuximab-resistant KRAS mutated colorectal cancer cells via trifluorothymidine incorporation into DNA. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B86.