The objective of the present study was to investigate the effects of polo‑like kinase 1 (PLK1) and the phosphorylation of human cell division cycle protein 14A (Cdc14A) by PLK1 on β‑cell function and cell cycle regulation. Mouse β‑TC3 cells were incubated with small interfering RNA (siRNA) to knock down the expression of PLK1. Cell cycle analysis was performed using flow cytometry, and cell proliferation and apoptosis was determined. Insulin secretion was evaluated by a radioimmunoassay under both low and high glucose conditions. Mouse β‑TC3 cells were transfected with a wild type or a non‑phosphorylatable Cdc14A mutant (Cdc14AS351A/363A; Cdc14AAA) to investigate whether the phosphorylation of Cdc14A is involved in cellular regulation of PLK1 under high glucose conditions. It was found that PLK1 siRNA significantly promoted cellular apoptosis, inhibited cell proliferation, decreased insulin secretion and reduced Cdc14A expression under both low and high glucose conditions. Cdc14A overexpression promoted β‑TC3 cell proliferation and insulin secretion, while Cdc14AAA overexpression inhibited cell proliferation and insulin secretion under high glucose conditions. PLK1 siRNA partially reversed the proliferation‑promoting effects of Cdc14A and further intensified the inhibition of proliferation by Cdc14AAA under high glucose conditions. Similarly, Cdc14A overexpression partially reversed the insulin‑inhibiting effects of PLK1 siRNA, while Cdc14AAA overexpression showed a synergistic inhibitory effect on insulin secretion with PLK1 siRNA under high glucose conditions. In conclusion, PLK1 promoted cell proliferation and insulin secretion while inhibiting cellular apoptosis in β‑TC3 cell lines under both low and high glucose conditions. In addition, the phospho‑regulation of Cdc14A by PLK1 may be involved in β‑TC3 cell cycle regulation and insulin secretion under high glucose conditions.