There is a growing body of detailed research demonstrating that intermittent fasting is essentially a cleansing activity in terms of health. Especially since its applications that exceed 16h trigger autophagy, it continues its effect on all tissue and organ systems after the regeneration movement that starts at the cellular level. Similarly, it continues to be better understood with each passing day that the gut microbiota (GM) has many positive effects on all tissue and organ systems. Although the GM is affected by many different parameters, dietary habits are reported to be the most effective factor. Therefore, it is important to investigate the effects of different preferred fasting practices on the GM, which has numerous health benefits. Pointing out this situation, this study aims to determine the effects of 18-h intermittent fasting for 5weeks on the shaping of GM. A 12-month-old male Wistar rat was chosen as the model organism in the study. At the end of the application, the metagenome was applied to the cecum content of the intestinal tissue collected from the sacrificed animals. Intermittent fasting practice led to an increase in alpha diversity, which expresses a significant bacterial diversity, the stabilization of Firmicutes and Bacteroidetes ratios (F/B), and the reshaping of the values with the highest prevalence in all stages of the classification, especially in the family, genus, and species care. Analysis results showed that the preferred intermittent fasting program helps balance the GM composition. This study is an important example showing the strong positive link between intermittent fasting and GM.