The John Cunningham virus (JCV) infects a large proportion of the population worldwide and may cause progressive multifocal leukoencephalopathy upon immunodeficiency. Recent reports provided evidence of its oncogenetic role in malignancies. In this study, JCV was examined by targeting T antigen, viral protein and agnoprotein in paraffin-embedded or frozen gastrointestinal carcinomas and paired non-neoplastic mucosa (NNM) samples by nested-PCR followed by Southern blot analysis. In addition, the expression of JCV T antigen, ki-67, caspase-3, p53, Rb and β-catenin was studied by immunohistochemistry on tissue microarrays. The positive rate of JCV T antigen was higher in paraffin-embedded gastrointestinal carcinomas compared to adjacent NNM by nested-PCR followed by Southern blot analysis (36.9 vs. 16.9%, P<0.05), while there was no difference in other viral oncogenes regardless of whether they were paraffin-embedded or frozen samples. Immunohistochemically, T antigen was detectable in 9.6% (13/135) of carcinoma cases, which was higher than its positive rate in NNM (0.8%, 1/126, P<0.01). However, the genomic JCV DNA existence or its T antigen expression was not correlated with age, gender, tumor size, histological types, lymph node metastasis, expression of ki-67, caspase-3, p53, Rb and β-catenin of gastric carcinomas (P>0.05). In conlusion, JCV T antigen may be involved in gastrointestinal carcinogenesis as an oncogene in China.