Serum amyloid A (SAA) proteins are acute-phase proteins and are classified into multiple isoforms; however, the biological functions of each SAA isoform are not fully understood. In this study, to clarify the roles of SAA3 in the intestine, we characterized mRNA expression in mouse colonic epithelial CMT-93 cells treated with rotavirus, Toxoplasma, Staphylococcus aureus, and Escherichia coli, as well as lipopolysaccharide (LPS) and recombinant murine SAAs (rSAAs). E. coli together with LPS, but not the other pathogens, enhanced SAA3 mRNA expression. The mRNA expression of SAA3 by dead E. coli was higher than that by living E. coli, and the mRNA expression by E. coli and LPS increased in a dose-dependent manner. In contrast, mRNA expressions of SAA1 and/or SAA2 were not stimulated by any of the treatments. In comparisons of cell treatments with rSAA1 or rSAA3, rSAA3 significantly up-regulated the mRNA expression of mucin 2 (MUC2), a major component of the mucus layer of the intestines that acts as an epithelial cell barrier against pathogens, while MUC2 mRNA expression was not significantly increased by E. coli and LPS. Furthermore, treatment with rSAAs intensively induced tumor necrosis factor-α mRNA expression. These results suggest that SAA3 plays a role in host innate immunity in the colon by up-regulating MUC2 mucin production, which builds a physiological barrier of colonic epithelia against bacterial invasion.