Colorectal cancer is a life-threatening and prevalent type of cancer. However, a number of current treatments have serious side effects, which increase the need for alternatives. Non-steroidal anti-inflammatory drugs have potential chemopreventive capabilities. The present study aimed to confirm this, as well as to investigate potential pathways and reasons for this trait. To accomplish this, cancerous Colo320 and healthy CCD-18 cells were treated with various concentrations of naproxen sodium (NS). A caspase-3 assay revealed a statistically significant increase in caspase-3 activity in Colo320 cells (300%; P<0.01), but not in CCD-18 cells. This chemical was also associated with a significant decrease in Colo320 cell survival (-72.888%; P<0.01), but not CCD-18 cell survival. Furthermore, NS was found to significantly decrease the migration of Colo320 cells (86.58%; P<0.01). Finally, RNA sequencing of cells treated with NS revealed the statistically significant downregulation of the mucin 5B, oligomeric mucus/gel-forming, S100 calcium binding protein A9 and mucin 5AC, oligomeric mucus/gel-forming genes, which are upregulated in colorectal cancer and are known to contribute to cancer proliferation, stemness and drug resistance. These novel biological pathway results were further confirmed using ELISAs. The present study identified a novel molecular mechanism of the anti-colorectal cancer activity of NS.