As the important component of humus, fulvic acids (FA) have a good antidiarrhoeal effect on animals and humans, and have been worldwide used in animal husbandry and even clinical practice for a long time. Due to the extremely complex chemical composition and structure of FA, the material basis and mechanism of its antidiarrhoeal activity have not been fully elucidated. In this study, we used ultrafiltration technique to fractionate this heterogeneous mixture into a series of relatively uniform fractions. The main structural features of FA and its fractions were characterized, and at the same time their antidiarrhoeal activities on drug-induced diarrhoea model mice were evaluated and the collagen content in the intestine of mice were determined. Through contrastive study of the relative variations between structure characteristics and antidiarrhoeal activities with the change of molecular weight, we found that the oxygen-containing functional groups especially phenolic hydroxyl groups, molecular weight distribution, colloidal properties and astringency were the material basis of the antidiarrhoeal activity. Fulvic acid substances had a dual antidiarrhoeal mechanism acting on the intestinal mucosa. The components with low molecular weight (< 5 K) mainly acted on the inside of intestinal mucosa and the components with high molecular weight (> 5 K) acted on the surface, and they could simultaneously exert the antidiarrhoeal effects.