Sesaminol triglucoside (STG), the most abundant lignan glycoside existing in sesame cake/meal, has exhibited various biological activities. However, little information about its in vitro fermentation with intestinal microbiota is available. Therefore, the effect of STG from sesame cake on the fermentation of human fecal microbiota was evaluated. First, high-purity STG was successfully prepared from defatted sesame cake by extraction with 80% ethanol and simple purification procedures of polyamide column chromatography and Toyopearl HW-40S column chromatography. Then the influence of STG on intestinal microbiota was conducted by monitoring bacterial populations and analyzing the concentrations of short-chain fatty acids (SCFA). We found that STG could significantly induce an increase in numbers of Lactobacillus - Enterococcus group and Bifidobacterium in fermentation in vitro with human fecal microbiota, while it did not stimulate the bacterial growth of Eubacterium rectale - Clostridium coccoides group, Clostridium histolyticum group, and Bacteroides - Prevotella group. Furthermore, it was found that concentrations of formic, acetic, propionic, and butyric acids in STG culture increased significantly during the fermentation, and its total SCFA concentration was relatively higher than those of the control and glucose cultures at 6 and 12 h fermentation. Our findings provided further evidence for the importance of human intestinal bacteria in the bioactivity of STG and its metabolites in the maintenance of human health.