Background: Lung cancer is a leading cause of cancer-related death worldwide. Previously we demonstrated that polyphyllin I (PPI), a bioactive component extracted from Paris polyphylla, inhibited the growth of non-small cell lung cancer (NSCLC) cells through the SAPK/JNK-mediated suppressing p65, DNMT1 and EZH2 expressions. However, the molecular mechanism underlying anti-lung cancer effect by PPI still remain elusive. Purpose: In this current study, we further explored the molecular mechanism underlying the anti-lung cancer effect of PPI. Methods: MTT, Cell-Light (TM) EdU DNA cell proliferation and colony formation assays were used to measure cell growth. Western blot were used to examine protein levels of c-Jun and p21. The expression level of long non-codingth RNA HOX transcript antisense RNA (HOTAIR) was measured by qRT-PCR. The p21 promoter activity was measured by Dual-Luciferase Reporter Assay System. The transient transfection experiments were used to silence and overexpression of c-Jun, p21 and HOTAIR. Tumor xenograft and bioluminescent imaging experiments were carried out to confirm the in vitro findings. Results: We showed that PPI suppressed growth of NSCLC cells. Mechanistically, we observed that PPI reduced expression of HOTAIR, while increased transcription factor c-Jun protein levels. Additionally, PPI also induced protein expression and promoter activity of p21, a cyclin-dependent kinase inhibitor. While exogenously expressed HOTAIR showed no effect on c-Jun levels, silencing of c-Jun significantly reversed the PPI-inhibited HOTAIR expression. Moreover, excessive expressed c-Jun further enhanced PPI-inhibited HOTAIR expression and PPI-induced p21 protein levels. Intriguingly, overexpression of HOTAIR and silencing of c-Jun overcame the PPI-induced p21 protein and promoter activity. Finally, silencing of p21 neutralized the PPI-inhibited cell proliferation. Similar results were also found in one xenograft mouse model. Conclusion: Our results demonstrate that PPI inhibits growth of NSCLC cells through regulation of HOTAIR and c-Jun expressions, which lead to induction of p21 gene. The interactions among HOTAIR, c-Jun and p21 regulatory axis converge in the overall anti-lung cancer effect of PPI. This study unveils an additional new mechanism for the anti-lung cancer role of PPI.