Cyasterone was demonstrated potential inhibition effect in mouse skin carcinoma cells in published report. However, the molecular mechanisms of the cyasterone on cells remain unknown. Herein, we investigated the effects of cyasterone-induced apoptosis in A549 and MGC823 cells in vitro. MTT assay showed that cyasterone caused a significantly decreasing of the proliferation of A549 and MGC823 cells in a time-and dose-dependent manner with IC₅₀ values of 38.50±3.73μg/mL on A549 cells and 32.96±1.24μg/mL on MGC823 cells at 48h, respectively. Hoechst staining and TUNEL staining results indicated the quintessential apoptosis features in immunofluorescence image. Apoptosis and cell cycle were determined by flow cytometry. Cyasterone treatment triggered inhibition of epidermal growth factor receptor- phosphatidylinositol 3 kinase/protein kinase B (EGFR-AKT) signaling pathways and activation of P38 pathways. Furthermore, cyasterone inhibited MGC823 cells xenografted tumor growth in vivo with few changes in body weights. In conclusion, our findings provide the evidence that cyasterone inhibits growth of A549 and MGC823 cells, via regulating EGFR signaling pathway. Our results indicated that cyasterone, a natural EGFR inhibitor, maybe a promising anti-cancer agent.