In this study, the hypothesis that food-derived opioid peptides besides β-casomorphin 7 might modulate the production of mucin via a direct action on epithelial goblet cells was investigated in HT29-MTX cells used as a model of human colonic epithelium. Seven milk whey or casein peptides, a human milk peptide, and a wheat gluten-derived peptide with proved or probable ability to bind μ- or δ-opioid receptors were tested on the cell culture. Significantly increased secretion of mucins was found after exposure to six of the assayed peptides, besides the previously described β-casomorphin 7, as measured by an enzyme-linked lectin assay (ELLA). Human β-casomorphin 5 and α-lactorphin were selected to study the expression of mucin 5AC gene (MUC5AC), the HT29-MTX major secreted mucin gene. α-Lactorphin showed increased expression of MUC5AC from 4 to 24 h (up to 1.6-fold over basal level expression), although differences were statistically different only after 24 h of exposure. However, this increased expression of MUC5AC did not reach significance after cell treatment with human β-casomorphin 5. In conclusion, six food-derived peptides have been identifed with described or probable opioid activity that induce mucin secretion in HT29-MTX cells. Concretely, α-lactorphin is able to up-regulate the expression of the major secreted mucin gene encoded by these cells.