In the present study, AIB1siRNA‑loaded polyethyleneimine (PEI)/heparin/Ca2+ nanoparticles (NPs) were successfully prepared and evaluated for their efficacy in lung cancer cells. The results demonstrated that the PEI and heparin complex reduced the toxic effect in cancer cells while maintaining its transfection efficiency. A nanosized particle of ~25 nm was formulated and siRNA was demonstrated to possess excellent binding efficiency in the particles. Confocal microscopy revealed that fluorescein‑labeled (FAM)‑small interfering (si)RNA dissociated from the HA‑PEI/heparin/Ca2+/siRNA (CPH‑siH) NPs and exhibited maximum fluorescence in the cytoplasm, which was important in elucidating its post‑transcriptional activity. CPH‑siH NPs exhibited a typical concentration‑dependent toxicity in cancer cells. Blank PEI/heparin/Ca2+ did not induce any toxicity in cancer cells, indicating its safety and lack of side effects. CPH‑siH (100 nm) induced the maximum apoptosis of cancer cells with nearly ~35% of cells in the early and late apoptosis stages. The expression of the nuclear receptor coactivator 3 (NCOA3, also known as AIB1) protein was knocked down in a concentration‑dependent manner, demonstrating the potent activity of AIB1siRNA in cancer cells. Together, these results indicated that HA‑PEI/heparin/Ca2+ NPs may be a promising carrier for the anticancer activity of AIB1siRNA in lung cancer cells.