A fraction (designated as U3EE) with molecular mass under 3 kDa from the water extract of the earthworm Eisenia fetida was prepared and its effects on mammalian digestive enzymes were examined. U3EE itself showed no relevant enzyme activities. However, it increased the activities of trypsin, alpha-chymotrypsin, and lipase, while decreased those of a-amylase and alpha-glucosidase. The trypsin activation and alpha-amylase inhibition were analyzed precisely by enzyme kinetics. The former was solely dependent on increase in the molecular activity k(cat) without any change in the Michaelis constant K-m, and the latter was mainly dependent on decrease in K-m with a slight change in k(cat). Effects of the treatments of U3EE with freezing-and-thawing, heat, acid, and hexane were examined. The treated U3EE showed the effects on the enzymes with the same potencies as those provided by the non-treated one, indicating that U3EE was enough stable toward the harsh treatments to hold the modulating effects on the enzyme activities. U3EE was also shown to be highly hydrophilic. The results obtained in this paper suggested that U3EE could be applicable as a novel constituent for pharmaceuticals and functional foods.