The aim of the present study was to investigate the expression of keratin 20 (KRT20) and placenta specific 8 (PLAC8) in gastrointestinal (GI) cancer with various differentiation phenotypes. The present study retrospectively investigated archived formalin‑fixed paraffin‑embedded tissue samples from 12 patients at different stages of GI cancer [four with gastric cancer, four with pancreatic cancer and four with colorectal cancer (CRC)]. The stages were pre‑determined, according to differentiation phenotypes, by a pathologist of the Department of Pathology at Sijhih Cathay General Hospital. KRT20 and PLAC8 expression levels were assessed using immunohistochemistry. The CRC cell lines SW620 and Caco‑2 were used to assess interactions between KRT20 and PLAC8 via reverse transcription‑quantitative PCR. PLAC8 and KRT20 expression was observed consistently only in the well‑differentiated CRC tissue samples. Low KRT20 expression levels were observed in the PLAC8 knockdown SW620 cells. In addition, there was a positive association between PLAC8 and KRT20 expression in the differentiated Caco‑2 cells. According to the results of the present study, the differentiation status of GI cancer influenced KRT20 expression, particularly in CRC, which may explain why patients with well‑differentiated CRC display better clinical outcomes. Therefore, the prognostic significance of KRT20 and PLAC8 may be particularly crucial for patients with CRC displaying a well‑differentiated phenotype.