Abstract: Objective: To investigate the effect of Chinese herbal medicine Polyphyllin Ⅰ (PPⅠ) on the cell growth inhibition of lung cancer and its molecular mechanisms. Methods: MTT, Wound healing assay and cell cycle assay were performed to detect the effects of PPⅠ on the cell growth, migration and cell cycle of lung cancer. Western Blot analysis was used to determine the protein expression of transcriptional regulator Sp1 and integrin-linked kinase ILK. qPCR analysis was used to detect the expression of miR-542-3p and ILK mRNA. Overexpression plasmids or siRNAs of ILK and Sp1 were transfected by using cell transient transfection technique. Dual-luciferase reporter assay was performed to measure the ILK promoter activity. Results: The results showed that PPⅠ significantly inhibited the cell viability of A549 and PC9 cells in a time and dose dependent manner. Western Blot results confirmed that PPⅠ down-regulated the expression of ILK protein. Silencing of ILK inhibited cell viability and migration, and arrested the cell cycle in S phase. Conversely, overexpression of ILK promoted the cell viability and reversed the growth inhibitory effect of PPⅠ on A549 and PC9 cells. miR-542-3p significantly suppressed the expression of ILK protein through directly binding to the 3’-UTR of ILK mRNA. PPⅠ could effectively increase miR-542-3p expression a t the same time. On the other hand, the protein expression of Sp1, a transcription regulated factor bound to ILK promoter region, was significantly down-regulated by PPⅠ. Silencing of Sp1 inactivated the ILK promoter activity and decreased the expression of ILK protein. Overexpression of Sp1 effectively reversed the effect of PPI-reduced expression of ILK protein. Conclusion: PPⅠ significantly inhibited the cell growth of lung cancer by decreasing the ILK protein expression through increasing the level of miR- 542-3p and down-regulation of Sp1.