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Objective: To study the effect of mechanism of high glucose on apoptosis and cell cycle arrest of islet β cells via p27 pathway. Methods: Islet INS-1 cells were cultured and divided into groups. The control group was treated with ordinary medium, the high glucose group was treated with high glucose medium containing 25mmol/L glucose, the high glucose+si-NC group was treated with high glucose medium and transfected with NC siRNA, and the high glucose+si-P27 group was treated with high glucose medium and transfected with p27 siRNA. After 24 hours treatment, MTS assay was used to detect the cell viability A490, TUNEL assay was used to detect apoptosis rate, flow cytometry was used to detect the cell cycle distribution and western blot was used to detect the expression levels of P27, caspase-8 and cyclinD1. Results: Compared with those in the control group, the A490, the ratio of S phase and G2/M phase as well as the expression level of CyclinD1 decreased, while the apoptosis rate, the ratio of G0/G1 phase as well as the expression levels of P27 and caspase-8 increased in the high glucose group (P0.05); compared with those in the high glucose group and high glucose+si-NC group, the A490, the ratio of S phase and G2/M phase as well as the expression levels of cyclinD1 increased, while the apoptosis rate, the ratio of G0/G1 phase as well as the expression levels of p27 and caspase-8 decreased in the high glucose+si-P27 group (P<0.05). Conclusion: The apoptosis and cell cycle arrest induced by high glucose are related to P27 pathway activation.