Cancer stem cells (CSCs) are notoriously drug resistant and well known for their ability to undergo self-renewal and differentiation into more mature cancer cells, as well as for their tumour-initiating ability from a relatively small number of cells. There remains a notable dearth of investigations into the exact role and functions of isolated CSC populations. This study developed methods to selectively enrich CSC populations for drug targeting. SW480 and CT26 parental wild-type (WT) colorectal cancer cells were transfected with a vector encoding the octamer-binding transcription factor 4 (OCT4) promoter site regulating expression of enhanced green fluorescent protein (GFP). The most highly positive OCT4-GFP cell population after extensive rounds of sorting (the top ~1%–5%) could be further enriched by intermittent cycling involving alternating conditions of growth (between normoxia and anoxia). Cycled cell populations were examined by immunoblotting with OCT4 antibody and immunofluorescent staining for levels of OCT4 protein as a CSC marker. CT26 WT and enriched CT26 OCT4 cells were then injected intraperitoneally into BALB/c mice to compare their tumour-initiating capacity as CSCs. Celecoxib was tested for repurposing and its ability to inhibit the CT26 WT and CT26 OCT4-GFP cell lines from forming colorectal tumours in vivo. CT26 OCT4-GFP and SW40 OCT4-GFP showed significantly higher levels of OCT4 expression when compared with WT cells, based on Western blotting and immunofluorescence staining. The highly enriched CT26 OCT4-GFP CSC population produced significantly greater tumour numbers with larger sizes than did the CT26 WT inoculated mice. However, colorectal tumours formed by either cell types were significantly decreased (~50%) in numbers and volumes by celecoxib treatment. Significant levels of red blood cells were present in the peritoneal cavities of mice with the untreated colorectal tumours but greatly inhibited peritoneal angiogenesis was noted in the celecoxib-treated mice. Using these model systems for study will ensure that the role of CSC-enriched populations in tumour growth and metastasis and their therapeutic targeting can now be effectively conducted. The findings also support the potential for celecoxib repurposing and use in chemosensitising colorectal cancer cells, thereby rendering them more susceptible to standard chemotherapies, such as doxorubicin and 5-fluorouracil.