Awamori moromi vinegar (AMV) contains essential amino acids and citric acid; however, its peculiar flavor prevents its acceptance as a functional food material. In a previous study, a fermented product of AMV (FP-AMV) was prepared using Lactobacillus plantarum K-3 to resolve the peculiar flavor of AMV, and its possibility to improve lipid metabolism through an approach to gut microbiota was suggested. In this study, using in vitro and in vivo experiments, it was aimed to determine whether FP-AMV could be used as a prebiotic to improve gut microbiota. The in vitro prebiotic assay showed increased turbidity for eight lactic acid bacteria and five bifidobacteria with the addition of FP-AMV, suggesting the comprehensive bacterial growth-promoting effect of FP-AMV. In contrast, the growth of Clostridium perfringens was greatly suppressed by FP-AMV. Therefore, an animal experiment was conducted to investigate the relationship between FP-AMV ingestion and the gut microbiota. Gut microbiota analysis of fecal samples in animal experiments proved that FP-AMV induced not only an increase in the prevalence of probiotic species, such as Lactobacillus and Bacteroides, but also a decrease in the prevalence of pathogenic species, such as Clostridium, in the gut microbiota of male C57BL/6JJcl mice. These results suggest that FP-AMV contributes to the improvement of the gut microbiota and the gut environment. Thus, it can be used as a potential prebiotic food.