Colorectal carcinoma is a cosmopolitan type of cancer with a poor prognosis, motivating seeking novel strategies to prevent disease development and progression. The poor prognosis is attributed to the severe toxic side effects of the current therapeutic regimes. Hence, novel less toxic treatment strategies are urgently warranted. Berberine is a natural compound with several biological and pharmacological properties, including anti-fungal, anti-diabetic, cardioprotective effects. Some reports showed that berberine inhibits cell growth by inducing cell cycle arrest and promotion of apoptosis in cancer cells. Importantly, the anticancer potential of berberine in colorectal cancer has not been previously investigated. Hence, this work aims to investigate whether berberine possesses anticancer properties against colorectal HCT116 cancer cells. The potential effect of berberine on cell cycle regulation and apoptosis will also be deeply investigated. This work was conducted using the more physiological 3D spheroid culture model that mimics better the impact of the tumor microenvironment as well as the cell-cell interaction in the cellular response to therapy. When compared to the previous studies, this work will explain the mode of action of berberine in more physiological conditions that better mimics the in vivo situation. To achieve the goal of this work, spheroid growth assay, as well as proliferation assay, were performed. Spheroid cell suspensions were further investigated using flow cytometry to assess the cell cycle distribution of cells upon berberine application. BrdU immunostaining was performed to elucidate the S-phase fraction of cells. The proliferation potential and the level of apoptosis were also investigated by Ki67 and Annexin V labeling, respectively. The results showed that berberine attenuated tumor spheroid growth and limits the proliferative capacity of HCT116 cells. This could be attributed to the berberine-mediated G1-phase cell cycle delay. The S-phase fraction of cells was significantly decreased upon berberine application. Unexpectedly, berberine did not induce a significant difference in the % of apoptotic cell fraction of cells as compared to the controls. Collectively, these results suggest that berberine possesses an anti-tumor efficacy in 3D culture preparations via modulating the cell cycle progression. Specifically, berberine induces G1-phase cell cycle delay and decreases the S-phase fraction of cells. Thus, it limits the proliferative capacity of cells. Also, berberine did not induce programmed cell death in the HCT116 spheroids.