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The aim of this study was to investigate the effect of hexagonal selenium nanoparticles modified by SiRNA (HSNM-SiRNA) to inhibit epidermal growth factor receptor (EGFR) signaling in Human non-small-cell lung cancer (NSCLC). After synthesis, HSNM-SiRNA and HSNs were separately exposed to NSCLC cell lines (A549, H1299, H520, and H1975), and incubated for 6 h at 37 °C. Next, the expression of NFKB, MYC, STAT, ELK1, and GAPDH was evaluated by western blot and real-time PCR. The percentage of apoptotic cells and cell cycle progression were measured when exposed to HSNM-SiRNA and HSNs. Both western blot and real-time PCR results showed that HSNM-SiRNA could down-regulate the expression of all EGFR signaling genes. The percentage of apoptotic cells was significantly increased in all cell lines when exposed to HSNM-SiRNA (P > 0.05). HSNM-SiRNA in A549 and H1299 cells significantly increased the proportion of cells in G1/G0 phase and significantly decreased the proportion of cells in S phase.