Background: Trifluridine (FTD) is an active cytotoxic component of TAS-102, and incorporation of tri-phosphorylated FTD into DNA confers its anti-tumor effect. Recent reports suggest that decreased expression of human equilibrative nucleoside transporter (hENT) and thymidine kinase 1 (TK-1) results in decreased nuclear intake of FTD. We tested whether gene polymorphisms involved in FTD metabolism are associated with outcomes in patients with refractory metastatic colorectal cancer (mCRC) treated with TAS-102. Methods: We analyzed genomic DNA extracted from 104 blood samples of two different cohorts: an evaluation set of 52 patients receiving TAS-102 (median age 61 years, male 44%, median follow-up time, 6.4 months), and a control group of 52 patients receiving regorafenib without history of TAS-102 treatment (median age 64 years, male 44%, all patients deceased). Single nucleotide polymorphisms (SNPs) of genes in FTD metabolism (TK-1, hENT-1) were analyzed by PCR-based direct sequencing. The candidate SNPs were selected by their frequency and potential function. Associations between selectedSNPs and PFS and OS were evaluated by Kaplan-Meier and log-rank tests in the overall population. Cox proportional hazard regression model was used in multivariate analyses. Results: In univariate analysis, median PFS for patients with any hENT-1 rs760370 G allele was 3.5 vs. 2.1 months for those carrying the A/A genotype (HR 0.44, 95%CI: 0.23-0.83, p = 0.004). Median OS was 8.7 and 5.3 months in the two groups, respectively (HR 0.27, 95%CI: 0.10-0.70, p = 0.003). Among patients with any hENT-1 rs9394992 T allele, median PFS was 3.4 vs. 1.9 months for those with the C/C variant (HR 0.48, 95%CI: 0.25-0.91, p = 0.011). Median OS was 8.7 vs. 4.4 months (HR 0.21, 95%CI: 0.08-0.51, p < 0.001) in the two groups, respectively. In multivariate analysis, hENT-1 rs760370 and hENT-1 rs9394992 genotypes remained significantly associated with PFS and OS. No significant differences were observed in the control group. Conclusions:hENT-1 germline polymorphisms may serve as predictive and prognostic markers in mCRC patients treated with TAS-102.