Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) re-expressed and promoted hollow spheroid (HS) formation beyond the invasion front of colorectal cancer. The aim of the present study was to clarify whether CEACAM1 cytoplasmic domain isoform balance and HS are associated with resistance to 5-fluorouracil (5FU).Two-dimensional (D) or 3D culture systems were employed to evaluate the effects of CEACAM1 cytoplasmic isoform balance and HS formation on the chemosensitivity of colorectal cancer cells to 5FU. The risk factors for postoperative recurrence were calculated based on the presence of HS and various clinicopathological characteristics in 82 patients with Stage III colorectal cancer who had undergone curative surgery followed by 5FU-based chemotherapy.CEACAM1-4L-transfected HT29 and CEACAM1-4L and 4S expressing parental LS174T cells had significantly higher resistance to 5FU in comparison with CEACAM1-4S- or vector control-transfected cells. In 3D culture, HS formation induced by CEACAM1-4L induced chemoresistance to 5FU, whereas the solid spheres formed in response to CEACAM1-4S were destroyed by 5FU treatment. HS was identified as an independent factor for recurrence of Stage III colorectal cancer after curative resection followed by 5FU-based chemotherapy. Kaplan-Meier survival curves demonstrated that patients with HS had lower recurrence-free survival rate.CEACAM1 long cytoplasmic domain isoform dominance and HS formation are phenotypes associated with chemoresistance to 5FU.