Activation of nuclear factor-kappa B (NF-kappaB) signalling is considered a crucial mechanism in the development of cancers. Caspase-associated recruitment domain 6 (CARD6) is a protein that activates NF-kappaB signalling evoked by RIP1, RIP2, Bcl-10 and MEKK. In this study, we analysed tissue expression of CARD6 protein in oesophageal squamous cell carcinoma (ESCC), gastric adenocarcinomas (GC) and colorectal adenocarcinomas (CRC). We analysed the expression of CARD6 protein in 58 ESCC, 100 GC and 103 CRC patients' tissues by immunohistochemistry using a tissue microarray (TMA) approach. We found CARD6 immunostaining in cancer cells of ESCC (41/58; 70.7%), GC (45/100; 45.0%) and CRC (81/103; 78.6%). In the GC, intestinal-type GC (77.8%) showed higher expression of CARD6 than diffuse-type GC (20.0%) and mixed-type GC (50.0%). By contrast, corresponding normal epithelial cells of oesophagus (0%), stomach (8.0%) and colon (5.0%) displayed lower frequencies of CARD6 immunostaining. The CARD6 immunostaining was observed in nucleus/cytoplasm (ESCC) or cytoplasm (GC and CRC). The CARD6 expression was evident from an early TNM stage (stage I). The increased expression of CARD6 in ESCC, GC and CRC tissues compared to their corresponding normal cells suggested that neoexpression of CARD6 might be related to activation of NF-kappaB pathway in the cancers and might play a role in the development of most types of gastrointestinal cancers.