Cervical cancer is the most common malignancy of the female reproductive tract, and the poor response to prophylactic vaccines and the toxicity of high?dose chemotherapeutic drugs have limited their clinical application. Spermidine, a natural polyamine detected in all eukaryotic organisms, exhibits functions that promote longevity in multiple model systems and may constitute a promising agent for cancer treatment. However, the potential effectiveness of spermidine in cervical cancer has not yet been fully elucidated, and the underlying molecular mechanisms remain unclear. In the present study, we aimed to assess the effects of spermidine on proliferation and apoptosis of HeLa cells (a cervical cancer cell line). Firstly, CCK?8 and flow cytometric assays revealed that spermidine reduced the proliferation of HeLa cells in a dose?dependent fashion by arresting the cell cycle at the S?phase. Secondly, flow cytometry incorporating Annexin?V?FITC/PI?staining revealed that spermidine promoted the apoptosis of HeLa cells, and western blot analysis revealed that spermidine activated autophagy. Finally, spermidine?activated autophagy mediated the inhibition of cell proliferation by spermidine and spermidine?induced apoptosis in HeLa cells. Collectively, these results revealed a novel function for spermidine in inhibiting cellular proliferation and inducing apoptosis of HeLa cells by activating autophagy, which may have important implications for the use of spermidine in cervical cancer therapy.