Colorectal cancer is a prevalent global health issue. In Australia, it ranks as the third most common newly diagnosed cancer, with around 15,000 new cases annually(1). Despite treatment advances, high incidence and mortality rates highlight the need for effective prevention and new therapies. Polyphenols, abundant in plant-based foods, have shown promise in inhibiting cancer cell growth and inducing apoptosis, offering a dietary approach to reduce cancer risk and improve outcomes. Whole-grain cereals like sorghum are recognised sources of phenolic compounds and can scavenge free radicals(2). This study aimed to evaluate the role of sorghum-derived polyphenols in modulating the major cancer development pathways. Also, the impact of processing techniques (cooking and fermentation) on sorghum polyphenols and cancer was evaluated. Polyphenols were extracted from the raw, cooked, fermented, and fermented-cooked sorghum flour samples(3). The phenolic content was measured using benchtop chemical assays including the DPPH radical scavenging assay and the ferric-reducing ability of plasma assay. UHPLC analysis coupled with Online ABTS characterised the polyphenols present in these extracts and provided their antioxidant activities. Using these extracts, a resazurin red cytotoxicity assay was performed on HT-29 colorectal cancer cells to determine the optimal concentrations for the downstream experiments. HT-29 cells were incubated with the black sorghum phenolic extracts (500 ug/mL and 2000 ug/mL) for 12 and 24 hours. Following this, the gene expression of several cancer regulatory genes (APC, KRAS, TTN, GLUT-1, HIF-1a and HIF-1b) was evaluated by qPCR. Treatment of HT-29 cells with raw sorghum phenolic extracts significantly (p < 0.05) upregulated APC and TTN genes at 12 and 24-hour time points and the KRAS gene at 24-hour time points compared to the control. This indicates the impact of sorghum-derived polyphenols on genome mutation and instability in cancer development pathways. Also, treatment at 500 ug/mL significantly (p < 0.05) upregulated the expression of GLUT-1 suggesting the impact on dysregulated cellular metabolism cancer development pathway. Processed sorghum phenolic extracts also significantly regulated KRAS gene expression. Overall, the results from this study showed that sorghum polyphenols modulate the expression of key cancer development pathway-associated genes in HT-29 cells. The findings underscore the potential of dietary polyphenols in cancer prevention and highlight the need for further research to optimise their use and understand their mechanisms of action in vivo.