Expression of gastrointestinal biomarkers MUC1, MUC2, MUC5AC, small-intestinal mucin antigen (SIMA), villin, and CDX2 has been studied in colorectal adenocarcinoma (CRC). Little is known, however, about their expression in small-intestinal adenocarcinoma (SIA). We immunohistochemically compared 30 SIAs with 48 CRCs for the expression of these biomarkers. The results showed that all 6 proteins were variably expressed in SIA, but the frequencies of expression were significantly lower than those for CRC with the exception of MUC1. Specifically, positive staining for MUC1, MUC2, MUC5AC, SIMA, villin, and CDX2 was observed in 16 (53%), 17 (57%), 12 (40%), 15 (50%), 20 (67%), and 18 (60%) of SIAs and 25 (52%), 43 (90%), 39 (81%), 45 (94%), 47 (98%), and 47 (98%) of CRCs, respectively. In addition, SIAs more frequently exhibited a focal staining pattern for MUC2, MUC5AC, SIMA, and villin, whereas more diffuse immunoreactivity was evident in CRCs. Focal staining for MUC1 and diffuse staining for CDX2 were common for SIAs and CRCs. Furthermore, poorly differentiated SIAs tended to express MUC1 more frequently when compared with well- and moderately differentiated SIAs. These observations further support the notion that SIA is immunophenotypically distinct from CRC despite their morphologic similarity.