Abstract The key techniques of preparing the active peptide from Perinereies aibuhitensis were investigated and the effects on human lung cancer A549 cells were also studied in this study. Firstly, the best enzymatic hydrolysis conditions were determined through the single-factor and orthogonal tests using the proliferation inhibition rate as the index. The enzymatic hydrolysates (1~3 ku) were obtained by ultrafiltration and further purified by ion exchange chromatography, gel filtration chromatography and preparative chromatography. Then, the lung cancer A549 cells were used to measure the proliferation inhibition of PAP by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The morphologic changes of cells were observed through inverted microscope, AO/EB fluorescent stain and Hoechst fluorescent staining. The results showed that the optimal enzyme was alcalase and the optimal hydrolytic conditions were as follows: the temperature was 55°C, pH value was 11, the solid-liquid ratio was 1:1, the hydrolysis time was 6 h, and the amount of enzyme was 300 U/g. An anti-cancer peptide (PAP) was then obtained after purification and identified as Ile-Glu-Pro-Gly-Thr-Val-Gly-Met-Met-Phe. However, our results also demonstrated that PAP suppressed the proliferation of A549 cells in a time- and dose-dependent manner and the morphological features of cells were changed in this study. Therefore, our findings suggest that PAP can inhibit the proliferation of lung cancer A549 cells and induces apoptosis.