Paneth cell differentiation may occur in colonic epithelial neoplasms. However, its significance and mechanism of development remains unclear. Human defensin 5 is a specific marker of Paneth cells and has been shown to represent one of the target genes of the Apc/beta-catenin/Tcf pathway. The aim of this study was to evaluate the frequency of Paneth cell differentiation in a variety of colonic neoplasms, and to investigate the role of human defensin 5 and beta-catenin in this process. The clinical and pathologic findings, including histologic evidence of Paneth cell differentiation and immunostaining for human defensin 5 and beta-catenin, were evaluated in 29 samples of nonneoplastic colonic mucosa, 18 hyperplastic polyps, 10 sessile serrated adenomas, 12 traditional serrated adenomas, 21 mixed polyps, 39 conventional adenomas, and 40 adenocarcinomas. Human defensin-5 and beta-catenin expression were evaluated for the location and degree of staining in all cell types (dysplastic and nondysplastic) and correlated with histologic areas of Paneth cell differentiation in all types of polyps. Histologic evidence of Paneth cell differentiation was observed in 15 conventional adenomas (38.5%) and 1 adenocarcinoma (2.5%) but not in other types of polyps. Human defensin-5 immunostaining was positive in the cytoplasm of all nonneoplastic Paneth cells and all neoplastic cells with Paneth cell differentiation. Human defensin-5 expression was noted in 0% of hyperplastic polyps, 10% of sessile serrated adenomas, 25% of traditional serrated adenomas, 33.3% of mixed polyps, 82.1% of conventional adenomas, and 17.5% of adenocarcinomas: human defensin 5 expression was significantly higher in conventional adenomas compared to all other groups (P < .01). Seventeen (53.1%) of 32 human defensin 5 positive conventional adenomas, 6 (86%) of 7 of human defensin 5 positive adenocarcinomas, and all human defensin 5-positive sessile serrated adenomas, traditional serrated adenomas, and mixed polyps did not show histologic evidence of Paneth cell differentiation. All mixed polyps (100%) that revealed human defensin 5 expression (7; 33.3%) revealed conventional dysplasia. In the positive mixed polyp cases, human defensin 5 was only positive in areas of conventional dysplasia. Of the 31 conventional adenomas with nuclear beta-catenin staining, 15 (48.4%) revealed histologic evidence of Paneth cell differentiation, and all of the neoplastic cells with Paneth cell differentiation showed nuclear beta-catenin staining, whereas nonneoplastic Paneth cells consistently showed a normal pattern of membranous beta-catenin staining. A strong topographical correlation was noted between human defensin 5 expression and nuclear beta-catenin expression in conventional adenomas and in conventional dysplastic epithelium of mixed polyps. Paneth cell differentiation is common in early colonic neoplasms that develop via the conventional adenoma-carcinoma carcinogenic pathway. Activation of Apc/beta-catenin/Tcf pathway may play a role in Paneth cell differentiation in human colonic neoplasms.