UNLABELLED: Colorectal cancer (CRC) remains as a major health problem with high lethality rate in the world. The innovate therapeutic strategies are essential in CRC management. The purpose of this research was to evaluate the effect of chitosan nanoparticle containing culture supernatant (CNP/Cj-sup) on genes involved in CRC signaling pathways. CNP/Cj-sup was fabricated via ionotropic gelation method. Dynamic light scattering and transmission electron microscopy (TEM) techniques were employed to characterize of the CNP/Cj-sup including the electrical charge, size distribution, and morphological properties. The loaded protein, released protein, and entrapment efficacy (EE) were assayed utilizing a BCA assay kit. After the evaluation of the viability of Caco-2 (colon adenocarcinoma) and HDF (human dermal fibroblasts) cells against CNP/Cj-sup by MTT assay, subsequently anti-tumor effect of CNP/Cj-sup on genes associated with CRC signaling pathways was assessed via real-time PCR method. The size dispersion of CNP/Cj-sup was 400.6 ± 24.4 nm with an electrical charge of + 4.5 mV. The loaded protein was calculated 1100 µg. The release rate of protein from CNP/Cj-sup was 78% at pH of 6.8 after 48 h, with EE of 74.62%. The viability of Caco-2 and HDF cells against CNP/Cj-sup (1100 µg + 0.05%) was measured 75.8 and 96.5%, respectively after 48 h. CNP/Cj-sup exhibited the highest efficacy in inhibiting the expression of oncogenes , and by to 0.06, 0.34, 0.14, 0.13, 0.08, and 0.14-fold ( value < .0001). Moreover, it led to a significant increase in the expression of the suppressor genes and by to 55.7 and 1.8- fold ( value < .0001). CNP/Cj-sup demonstrated the highest efficiency in suppressing and enhancing compared to CNP and Cj-sup. In conclusion, CNP/Cj-sup as an innovative potential anticancer agent, with the ability to modulate genes involved in CRC progression, represents a promising approach to CRC treatment. GRAPHICAL ABSTRACT: The effect of CNP/Cj-sup on different colorectal cancer signaling pathways.