Colorectal cancer (CRC) constitutes a major public health problem because of the high rate of morbidity and mortality. Chemotherapy and immunotherapy are the major and promising strategies for cancer patients including CRC; nevertheless, chemoresistance and immune escape limit the final efficacy of the above approaches. FERMT3 has proven to exert a critical role in the immune system and has contradictive effects on cancer progression. In this study, bioinformatics database analysis and clinical specimen detection both corroborated the downregulation of FERMT3 in CRC tissues and cells. Of interest, overexpression of FERMT3 suppressed CRC cell invasion and sensitized cells to 5-fluorouracil (5-FU) by reducing cell viability and increasing cell apoptosis and caspase 3 activity. Noticeably, FERMT3 upregulation enhanced natural killer (NK) cells activation by increasing secretions of interferon γ (IFN-γ) and tumour necrosis factor α (TNF-α) when NK cells were co-cultured with CRC cells. Importantly, upregulation of FERMT3 promoted NK cell-mediated killing of CRC cells. Mechanically, FERMT3 inhibited the aberrant activation of Wnt/β-catenin signalling and the subsequent programmed death-ligand 1 (PD-L1) expression in CRC cells. Moreover, knockdown of PD-L1 suppressed CRC cell invasion, 5-FU resistance and NK cells-mediated tumour killing. Additionally, reactivating the Wnt/β-catenin signalling with a specific WNT agonist CAS 853220-52-7 overturned the efficacy of FERMT3 overexpression against CRC cell invasion, 5-FU chemoresistance and cell susceptibility to NK cell-mediated cytotoxicity. Therefore, the current findings substantiate that FERMT3 elevation may attenuate CRC cell chemoresistance and NK cell-mediated immune response to tumour cells by inhibiting Wnt/β-catenin-PD-L1 signalling. Therefore, FERMT3 elevation may be a promising therapeutic approach to overcome chemoresistance and immune evasion in CRC.