The regulatory effect of dietary fiber complex (DFC) on the representative human gut microbiota and derived metabolites was explored. Faeces of 10 elderly people aged 65~75 years were collected, and in vitro fermentation of the DFC for intervention was performed according to gender. It was found that the intervention effect of the 2% DFC addition was the best, and the relative expressions of Escherichiacoil, Bacteroides, Bifidobacterium and Lactobacillus for the female group were 52.76%, 43.40%, 249.79% and 232.99%, respectively, of those in the control group. The relative expressions of of Escherichiacoil, Bacteroides, Bifidobacterium and Lactobacillus for the male group were 44.02%, 53.14%, 228.71% and 206.11%, respectively, of those in the control group. Among the significantly different gut flora metabolites, the relative abundances of isobutyrate, propionate and formic acid increased significantly (P<0.01), whilst the relative abundance of methionine, histidine, β-glucose, phenylalanine, threonine, alanine, aspartate, arginine and 1-methylhistidine decreased significantly (P<0.01) in the female group. In the male group, the relative abundance of isobutyrate, propionate, butyrate and formic acid increased significantly (P<0.01), whilst the relative abundance of histidine, β-glucose, phenylalanine, threonine and aspartate decreased significantly (P<0.01). The underlying metabolic pathways for both groups were histidine metabolism, and phenylalanine, tyrosine and tryptophan biosynthesis. The research results showed that DFC has a significant regulatory effect on human gut microbiota and derived metabolites, and the changes in the potential metabolic pathways are consistent across genders.