Background: Colorectal cancer (CRC) stands as one of the most prevalent cancer types and among the most frequent causes of cancer-related death globally. Acacia concinna (AC) is a medicinal and edible plant that exhibits a multitude of biological properties, including anticancer properties. This study aimed to investigate the impact of the AC extract on apoptosis induction and the underlying mechanisms associated with this effect in KRAS-mutated human colon HCT116 cells. Methods: The effect of AC extract on cell cytotoxicity was evaluated using MTT assay. Nuclear morphological changes were visualized with Hoechst 33342 staining, while mitochondrial membrane potential (MMP) was assessed via JC-1 staining. Flow cytometry was employed for cell cycle analysis, and intracellular ROS levels were determined using DCFH-DA staining. Results: The results showed that HCT116 cells exposed to AC extract showed reduced cell growth and prompted apoptosis, as indicated by an increase in chromatin condensation, apoptotic bodies, the sub-G1 apoptotic cell population, and disrupted MMP. Expression levels of apoptosis mediator proteins determined by Western blot analysis showed an increase in pro-apoptotic proteins (Bak and Bax) while decreasing anti-apoptotic proteins (Bcl-2, Bcl-xL, and Mcl-1), leading to caspase-7 activation and PARP inactivation. AC extract was also found to enhance intracellular reactive oxygen species (ROS) levels and stimulate endoplasmic reticulum (ER) stress. Furthermore, AC extract increases the phosphorylation of ERK1/2, p38, and c-Jun while downregulating PI3K, Akt, β-catenin, and their downstream target proteins. Conclusions: These results demonstrate that AC extract could inhibit cancer cell growth via ROS-induced ER stress associated with apoptosis and regulate the MAPK, PI3K/Akt, and Wnt/β-catenin signaling pathways in HCT116 cells. Therefore, AC extract may be a novel candidate for natural anticancer resources for colon cancer treatment.