A herbal alkaloid Berberine (Ber), used for centuries in Ayurvedic, Chinese, Middle-Eastern, and native American folk medicines, is nowadays proved to function as a safe anticancer agent. Yet, its poor water solubility, stability, and bioavailability hinder clinical application. In this study, we have explored a nanosized carbon nanoparticle—C₆₀ fullerene (C₆₀)—for optimized Ber delivery into leukemic cells. Water dispersions of noncovalent C₆₀-Ber nanocomplexes in the 1:2, 1:1, and 2:1 molar ratios were prepared. UV–Vis spectroscopy, dynamic light scattering (DLS), and atomic force microscopy (AFM) evidenced a complexation of the Ber cation with the negatively charged C₆₀ molecule. The computer simulation showed that π-stacking dominates in Ber and C₆₀ binding in an aqueous solution. Complexation with C₆₀ was found to promote Ber intracellular uptake. By increasing C₆₀ concentration, the C₆₀-Ber nanocomplexes exhibited higher antiproliferative potential towards CCRF-CEM cells, in accordance with the following order: free Ber < 1:2 < 1:1 < 2:1 (the most toxic). The activation of caspase 3/7 and accumulation in the sub-G1 phase of CCRF-CEM cells treated with C₆₀-Ber nanocomplexes evidenced apoptosis induction. Thus, this study indicates that the fast and easy noncovalent complexation of alkaloid Ber with C₆₀ improved its in vitro efficiency against cancer cells.