Colorectal cancer (CRC) remains a leading cause of cancer-related deaths worldwide, necessitating the development of novel therapeutic approaches. Carbon dots (CDs) have emerged as promising nanoparticles for biomedical applications due to their unique properties. Gallic acid (GA), an anticancer agent, is effective against various tumor types. This study explores the potential of gallic acid-derived carbon dots (GA-CDs) as an innovative anticancer agent against HCT-116 CRC cells, focusing on apoptosis signaling pathways. GA-CDs were synthesized using a one-pot hydrothermal method. Characterization was conducted using transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, and ultraviolet-visible (UV-vis) absorption spectroscopy. The cytotoxicity of GA and GA-CDs on HCT-116 cells was evaluated using the MTT assay at various concentrations over 24 and 48 h. Cellular uptake was assessed via fluorescence microscopy, and apoptosis was analyzed using acridine orange/propidium iodide (AO/PI) staining. Total RNA extraction followed by complementary DNA (cDNA) synthesis via reverse transcription-PCR was performed, and real time-PCR (Q-PCR) was conducted to examine the expression of apoptosis-related genes including3,, and2. Characterization confirmed the successful synthesis of spherical GA-CDs. GA-CDs exhibited dose- and time-dependent cytotoxicity, with IC50 values of 88.55 μg mlfor GA-CDs and 192.2 μg mlfor GA after 24 h. Fluorescence microscopy confirmed the efficient uptake of GA-CDs by HCT-116 cells. AO/PI staining showed a significant increase in apoptotic cell numbers after treatment with GA-CDs. Q-PCR analysis revealed overexpression of3 andgenes in GA-CD-treated cells, though no significant changes were observed in the expression of2 or the2 ratio. GA-CDs demonstrated potent anticancer properties by inducing apoptosis and reducing cell viability in HCT-116 cells. These findings suggest the potential of GA-CDs as a novel therapeutic agent for CRC treatment, warranting further investigation into their mechanism of action andefficacy.