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In this study, in vitro colonic fermentation and 16S rRNA high-throughput sequencing were used to explore the effects of the Cyperus esculentus dietary fiber on the production of short-chain fat acids (SCFAs) by the human gut microbiota and its structure. Soybean dietary fiber served as the control. The results showed that C. esculentus dietary fiber significantly promoted the production of SCFAs, but its effect was less pronounced than that of soybean dietary fiber. This study also found that high concentrations of dietary fiber significantly changed the diversity and richness of the gut microbiota, and the effects of the two dietary fibers were significantly different. C. esculentus dietary fiber significantly promoted the relative abundance of Firmicutes, Proteobacteria and Actinobacteria, but inhibited the growth of Bacteroidota. Its effect was slightly different from that of soybean dietary fiber. Moreover, compared with soybean dietary fiber, C. esculentus dietary fiber significantly had a stronger capacity to promote the growth and reproduction of Subdoligranulum, Bifidobacterium, Lactobacillus and Faecalibacterium, and significantly suppressed the growth of Prevotella and Bacteroides. This study highlights the potential of C. esculentus dietary fiber as a novel prebiotic.