Intestinal barrier dysfunction and mucus layer abnormalities are central features of inflammatory bowel disease, yet the epithelial mechanisms regulating goblet cell function remain incompletely understood. The TWEAK/Fn14 pathway is involved in intestinal inflammation, but its role in small intestinal epithelial responses, particularly in goblet cells, has not been fully clarified. In this study, we investigated TWEAK/Fn14 signaling using mouse small intestinal epithelial organoids. Fn14 was expressed in MUC2-positive cells in intestinal tissue, with staining preferentially observed toward the mucin-facing region. Fn14 staining was also observed in small intestinal epithelial organoids. TWEAK stimulation increased Tnf mRNA expression by approximately 1.56-fold and reduced Muc2 mRNA expression to approximately 66% of control, without markedly affecting epithelial proliferation or stem cell markers. TWEAK did not significantly alter the PAS-positive mucin area or AB-PAS-defined mucin composition, whereas PGM34 staining showed a non-significant tendency to increase. TNF treatment reproduced some of the TWEAK-associated changes, including reduced Muc2 mRNA expression and alterations in mucin-associated parameters, suggesting that TNF may contribute to some of the epithelial responses associated with TWEAK treatment. Treatment with an anti-TNF neutralizing antibody modified several TWEAK-associated responses, including changes in epithelial-associated gene expression and mucin-associated staining parameters. These findings suggest that TWEAK treatment is associated with goblet cell-related mucin responses in mouse small intestinal epithelial organoids, with possible involvement of TNF signaling.