The pathogenesis and etiology of pancreatic cancer remain to be fully elucidated; therefore, associated investigations are required to improve the outcome and prognosis of patients. In the present study, the effects of the overexpression of p16ink4a on the Wnt/β‑catenin signaling pathway were investigated in pancreatic cancer cell lines. Two pancreatic cancer cell lines, Bxpc‑3 and Miapaca‑2, characterized by low expression of p16ink4a, were transfected with the pc‑DNA3.0‑p16ink4a plasmid. After 24 h, Reverse transcription‑polymerase chain reaction and western blot analyses were performed to evaluate the expression of p16ink4a, β‑catenin, which is a key molecule in the Wnt/β‑catenin signaling pathway, c‑myc and cyclin D1, which are molecules downstream of β‑catenin. The expression of p16ink4a was significantly upregulated in the transfected cells. Consequently, the expression of β‑catenin was inhibited, whereas the expression levels of c‑myc and cyclin D1 were not altered significantly. The increased expression of p16ink4a may affect the activity of Wnt/β‑catenin signaling through modulation of the expression of β‑catenin. The results of the present study provide information for the future development of targeted treatments for pancreatic cancer.