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Introduction: Lung cancer is the most common type of cancer, causing worldwide mortality. Therefore, this study is necessary for continuing research into new effective and safe treatments. Recently, herbal medicines have been used for the treatment of various diseases such as cancer. This study aimed to investigate the potential anti-proliferative activity and investigate the mechanisms of hesperidin extract on the non-small lung cancer cells A549 and H460 vs. cisplatin via targeting the miR 34a/PD-L1/NF-κB signalling pathway. Material and methods: To determine the cytotoxic effects of the hesperidin extract on non-small lung cancer cells, sulphorhdamine B assay was performed. To show the inhibition of migration by hesperidin extract, wound healing assay was conducted. A quantitative polymerase chain reaction test was used to quantify the expressions of miR-34a, programmed cell death ligand-1 (PDL-1), epidermal growth factor receptor (EGFR), and P53 genes, which are involved in apoptosis pathway. Also, cell cycle assay was performed by using a flow cytometer. Results: The hesperidin extract could significantly inhibit proliferation of non-small lung cancer cells A549 and H460. Western blot assay demonstrated that hesperidin induced suppression of nuclear factor κB signalling pathway. The messenger RNA expression levels of MiR-34a and P53 were up-regulated significantly by hesperidin treatment, while the EGFR and P53 genes were down-regulated. The flow cytometer confirmed that cell cycle arrest occurred at the sub-G1 and G2 phases in A549 and H460, respectively. Conclusions: Our study demonstrated that hesperidin extract could significantly inhibit non-small lung cancer cell growth by induction of the apoptosis signalling pathway. Therefore, hesperidin might open novel strategies for effective and safe cancer treatment and reduce the adverse side effects of several chemotherapeutic treatments such as cisplatin. [ABSTRACT FROM AUTHOR]