Non-small cell lung cancer (NSCLC) is a malignant tumor that threatens the whole world, previous studies found that Polyphyllin II (PPII) can suppress NSCLC cells growth, exhibits significant antitumor activity. MicroRNAs (miRNAs) can regulate the expression of other genes and play a crucial role in the prevention and development of cancer. However, the miRNA portrait of ginsenoside PPII-treated NSCLC A549 cells has not yet been studied. In this work, miRNA-seq analysis was used to determine the changes in miRNA expression profile of NSCLC A549 cells PPII-treaded. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed on differentially expressed miRNA target genes. Then, predicted the target genes of miRNA and performed functional enrichment analysis on them. We identified 58 up-regulated and 24 down-regulated miRNAs displaying changes their expression in PPII treated A549 cells were greater than 2-fold. In addition, the expression levels of miRNAs were validated by real-time PCR. Therefore, this work has a certain promoting effect on the study of the anti-cancer mechanism of PPII in lung cells.