The global increase in prevalence of (pre-)diabetes demands immediate intervention strategies. In our earlier work, we demonstrated in vitro antidiabetic potential of a fermented beetroot product (PN39). Here, we examined the impact of PN39 on glucose tolerance and gut microbiota in C57BL/6J male mice and on prediabetic (PD) subjects' stool microbiota. In mice, high-fat diet (HFD) consumption for 9 weeks resulted in hyperglycemia and impaired glucose tolerance (GT) while concomitant consumption of PN39 and HFD (PN39+HFD) prevented GT impairment. Meanwhile, feeding the mice with HFD for 5 weeks to induce PD and later administering them with PN39 for 4 weeks (PD + PN39) neither improved fasting blood glucose nor GT. Relative to control groups, the gut microbiota of both PD mice and humans were characterized by decreased Clostridia UCG-014 and Lactobacilli as well as significantly altered gut microbial carbohydrate metabolism. Feeding PN39 together with HFD preserved Clostridia UCG-014 and Lactobacilli, increased short chain fatty acid production relative to mice fed with HFD only. Treating gut microbiota of PD subjects with PN39 however increased Clostridia UCG-014 and Lactobacilli populations and increased short chain fatty acids concentrations in the stools. In both mice and humans, PN39 treatment rectified the altered microbial carbohydrate metabolism observed in their PD counterparts. This suggests that the gut microbial modulatory effects of PN39 coupled with its capacity to regulate gut microbial glucose metabolism, likely played a role in preventing PD in mice receiving PN39+HFD. Taken together, our results indicate that PN39 could act as a potent antidiabetic functional food for preventing diabetes and its associated dysbiosis.