Accumulating evidence suggests that cancer cells with stem cell-like features have higher resistance to chemotherapeutic agents. Herein, we identified T-lymphoma invasion and metastasis-inducing protein-1 (TIAM1) as one of the Wnt-signaling associated genes which drives self-renewal and its expression is upregulated by cancer associated fibroblasts (CAFs). TIAM1 expression was assessed in resected colorectal cancer (CRC) tissues from 300 patients who did or did not respond to chemotherapy. siRNA and CRISPR/Cas9 was used to examine whether the inhibition of TIAM1 affects chemosensitivity of CRC. We demonstrate that stemness through Wnt signaling regulates chemosensitivity and this phenomenon occurs exclusively in cancer stem cells. Subsequently, we established patient-derived CAFs and tested whether the drug sensitivity of CRC cell lines is altered with CAF-derived conditioned medium. High-TIAM1 expression correlated significantly with poor prognosis of CRC patients, and was overexpressed in patients who did not respond to chemotherapy. We demonstrated that the inhibition of TIAM1 enhanced sensitivity to chemotherapeutic drugs and reduced tumor invasiveness in a series of experiments in vitro. Moreover, CAF-derived conditioned media increased stemness and chemoresistance in CRC cell lines through TIAM1 overexpression. In addition, we validated TIAM1 associated drug sensitivity using a xenograft model. We have demonstrated that TIAM1 is overexpressed in CRC tumors from patients who did not respond to chemotherapeutic drugs and levels of TIAM1 expression served as an independent prognostic factor. Mechanistically, CAFs enhanced CRC chemoresistance through TIAM1 overexpression. Collectively, these results suggest that TIAM1 regulates chemosensitivity in tumors and stroma and thus may be an attractive therapeutic target.