Abstract: Objective: To investigate the effect of argininosuccinate synthetase 1 (ASS1) on cell proliferation and apoptosis of pancreatic islet β cells. Methods: Small interference RNA was used to knockdown ASS1, and lentiviral vector to overexpress ASS1 in pancreatic islet β cells. 5‐ethynyl‐2′‐deoxyuridine (EdU) and cell counting kit-8 (CCK-8) to detect proliferation ability, Annexin V-PI flow cytometry and terminal dUTP nick-end labeling (TUNEL) to detect apoptosis. Western blot was used to examined protein expression of Bcl-2 (B-cell leukaemia-2), Bax (Bcl-2 Associated X Protein), Caspase-3, Cleaved Caspase-3, apoptosis inducing factor (AIF), nuclear proliferation antigen Ki67 and mammalian rapamycin target protein (mTOR). RT-qPCR was applied to detect mRNA expression of AIF, Ki67 and mTOR. Data between groups was compared with one-way ANOVA. Results: 1. Proliferation activity of pancreatic β cells detected by EdU and CCK-8 was lower when ASS1 was knockdown. and apoptosis of islet β cells examined by TUNEL method and flow cytometry was higher than that in NC group; 2. Apoptosis of β cells decreased when ASS1 was overexpressed. Meanwhile, increased AIF expression and decreased ratio of Bax/Bcl-2 and the activity of Caspase-3 were observed. Conclusion: Overexpressing ASS1 in pancreatic islet β cells may activate mTOR signaling pathway to promote cell proliferation. Pancreatic islet β cells with decreased ASS1 expression may initiate apoptosis through AIF pathway and inhibit the activities of Bax and Caspase-3 by negative feedback. In summary, ASS1 plays a role in pancreatic islet β-cell’s proliferation and apoptosis.