Cancer stem cells (CSCs) are the major culprits leading to a new level of complexity and the consequential therapy resistance and disease recurrence in colorectal cancer (CRC). This study focuses on the effect of long non-coding RNA (lncRNA) TPT1-AS1 and its associated molecules on the stemness maintenance of CRC stem cells. TPT1-AS1 was identified as a significantly upregulated gene in CRC using the GSE146587 dataset. Stem cells from CRC HCT116 and CACO2 cells were isolated. TPT1-AS1 was significantly highly expressed in the CSCs compared to non-stem cells. Downregulation of TPT1-AS1 reduced the stemness of the CRC stem cells. TPT1-AS1 recruited STAT1 to the promoter region of APC to suppress APC transcription. Further upregulation of STAT1 or downregulation of APC blocked the role of TPT1-AS1 silencing and restored the malignant behaviors of CSC stem cells. APC inactivated the Wnt/β-catenin pathway. Overexpression of STAT1 restored the levels of cyclin D1 and β-catenin in cells suppressed by TPT1-AS1 silencing. In summary, this work demonstrates that TPT1-AS1 recruits STAT1 to suppress APC transcription and increase the stemness of colorectal CSCs via Wnt/β-catenin activation.Keywords: APC; CSCs; Colorectal cancer; Long non-coding RNA TPT1-AS1; STAT1.