Colorectal cancer (CRC) is a malignancy of the colon or rectum. It is ranked as the third most common cancer in both men and women worldwide. Early resection permitted by early detection is the best treatment, and chemotherapy is another main treatment, particularly for patients with advanced CRC. A well-known thymidylate synthase (TS) inhibitor, 5-fluorouracil (5-FU), is frequently prescribed to CRC patients; however, drug resistance is a critical limitation of its clinical application. Based on the hypothesis that extract (CRE) can abolish this 5-FU resistance, we explored the efficacy and underlying mechanisms of CRE in 5-FU-resistant (HCT116/R) and parental HCT116 (HCT116/WT) cells. Compared to treatment with 5-FU alone, combination treatment with CRE and 5-FU drastically reduced the viability of HCT116/R cells. The cell cycle distribution assay showed significant induction of the G0/G1 phase arrest by co-treatment with CRE and 5-FU. In addition, the combination of CRE and 5-FU notably suppressed the activity of TS, which was overexpressed in HCT116/R cells, as compared to HCT116/WT cells. Our findings support the potential of CRE as an adjuvant agent against 5-FU-resistant colorectal cancers and indicate that the underlying mechanisms might involve inhibition of TS expression.