N-Acyl-homoserine lactones (AHL) are quorum-sensing molecules in bacteria that play important roles in regulating virulence gene expression in pathogens such as Pseudomonas aeruginosa. The present study compared responses between undifferentiated and differentiated Caco-2 cells to N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C₁₂-HSL). A low concentration of 3-oxo-C₁₂-HSL (30 μM) is sufficient to reduce viability accompanied by apoptosis via the suppression of phosphorylation by Akt in undifferentiated Caco-2 cells. The suppression of Akt phosphorylation appears specific in 3-oxo-C₁₂-HSL, because other AHLs did not influence the phosphorylation status of Akt. The reduced viability induced by 3-oxo-C₁₂-HSL was partially recovered by constitutively active Akt overexpression in undifferentiated Caco-2 cells. Since mucin is considered a vital component of the gut barrier, we investigated whether mucin protects cellular functions induced by 3-oxo-C₁₂-HSL in undifferentiated Caco-2 cells. The results showed that mucin protected undifferentiated Caco-2 cells from apoptosis induced by 3-oxo-C₁₂-HSL. 3-Oxo-C₁₂-HSL did not induce cell death in differentiated Caco-2 cells that expressed higher levels of mucin 3 (MUC3) than undifferentiated Caco-2 cells. In addition, 3-oxo-C₁₂-HSL promoted cell death in undifferentiated Caco-2 cells transfected with MUC3 siRNA and reduced MUC3 expression in undifferentiated Caco-2 cells. Therefore, MUC3 might be responsible for the survival of undifferentiated intestinal epithelial cells in the presence of 3-oxo-C₁₂-HSL through regulating Akt phosphorylation. In conclusion, 3-oxo-C₁₂-HSL might influence the survival of undifferentiated intestinal epithelial cells as well as interactions between these cells and pathogens.