Cancer stem cells (CSCs) have key roles in treatment resistance, tumour metastasis and relapse. Using colorectal cancer (CC) cell lines, patient-derived xenograft (PDX) tissues and patient tissues, here we report that CC CSCs, which resist chemoradiation, have higher SUMO activating enzyme (E1) and global SUMOylation levels than non-CSCs. Knockdown of SUMO E1 or SUMO conjugating enzyme (E2) inhibits CC CSC maintenance and self-renewal, while overexpression of SUMO E1 or E2 increases CC cell stemness. We found that SUMOylation regulates CSCs through Oct-1, a transcription factor for aldehyde dehydrogenases (ALDHs). ALDH activity is not only a marker for CSCs but also important in CSC biology. SUMO does not modify Oct-1 directly, but regulates the expression of TRIM21 that enhances Oct-1 ubiquitination and, consequently, reducing Oct-1 stability. In summary, our findings suggest that SUMOylation could be a target to inhibit CSCs and ultimately to reduce treatment resistance, tumour metastasis and relapse.