This study examined the effects of berberine, a bioactive alkaloid, on the apoptosis, proliferation, migration, and oxidative stress of DBTRG brain cancer cells and evaluated its potential when incorporated into a nanoparticle-mediated drug delivery system. DBTRG cells treated with 0.5, 1, 5, or 10 μg/mL of berberine for 48 h showed increased apoptosis through both intrinsic and extrinsic pathways, as evidenced by elevated annexin V+/propidium iodide- cells relative to untreated controls. Berberine effectively reduced cell proliferation by inducing cell cycle arrest at G1 and G2/M phases. It also inhibited cell migration by downregulating matrix metalloproteinases and modifying the cytoskeletal structure, and alleviated oxidative stress by enhancing antioxidant enzyme activity and lowering reactive oxygen species production. To overcome the limitations of berberine's low bioavailability, a nanoparticle-based delivery system was developed. The gold-collagen-berberine (Au-Col-BB) nanocarrier was characterized using UV-vis spectrophotometry, Fourier-transform infrared spectroscopy, dynamic light scattering, energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, and scanning electron microscopy. Au-Col-BB nanoparticles were engineered to enhance berberine's loading capacity and therapeutic efficacy. These nanoparticles entered DBTRG cells via endocytosis and progressed through the endosome-lysosome pathway, which significantly increased cellular uptake and therapeutic effectiveness. Annexin V/propidium iodide staining and cell cycle analysis demonstrated that Au-Col-BB nanoparticles promoted DBTRG cell apoptosis. The sub-G1 phase cell population increased by 19.4% ( < 0.001) compared to controls, while the S phase population decreased by 5.6% ( < 0.001), indicating enhanced apoptotic activity and reduced proliferation. In vivo analysis via retroorbital sinus injection of Au-Col-BB into BALB/c mice ( = 5) confirmed the nanoparticles' structural integrity and safety, as well as efficient accumulation in brain tissue. These findings underscore berberine's potential as an anticancer agent, particularly when delivered through a nanoparticle-based system to address the challenges of limited bioavailability and achieve targeted delivery to cancer cells.