Intermittent fasting is a nutritional strategy that focuses on when to eat, rather than what to eat. Although the effectiveness of intermittent fasting practices in many metabolic diseases is known, its effect on microbiota and its underlying mechanism has not yet been clarified. This study aimed to investigate the effect of Ramadan fasting, one of the intermittent fasting practices, on gut microbiota and fatty acid binding protein 4 (FABP4). The study involved 10 male volunteers, 6 of whom were overweight and 4 were obese. They fasted for an average of 14-15 hours daily from dawn to sunset during the 29-day Ramadan month between 23 March - 20 April 2023 and met the inclusion criteria. The participants' nutritional and physical activity status before and during Ramadan, as well as their anthropometric measurements before and after Ramadan, intestinal microbiota, transaminases, gamma-glutamyl transferase, C reactive protein, total cholesterol (C), high-density lipoprotein (HDL) C, low-density lipoprotein C, triglycerides (TG), and FABP4 levels, were evaluated within the scope of the study. The study found a statistically significant increase in both alpha and beta diversity in the intestinal microbiota following Ramadan fasting (p<0.05). The F/B ratio, Firmicutes phylum, Clostridia class, Clostridiales order, and Ruminococcaceae family exhibited statistically significant decreases, while the Bacteroidetes and Proteobacteria phyla, Bacteroidia, Alphaproteobacteria, and Erysipelotrichi classes, Bacteroidales, Erysipelotrichales, and Actinomycetales orders, Erysipelotrichaceae family and Prevotella genus, demonstrated statistically significant increases (p<0.05). Participants who achieved an average weight loss of 2.3±0.99 kg at the end of Ramadan showed a significant increase in HDL-C and a significant decrease in TG levels (p<0.05). Although FABP4 levels decreased after fasting, this difference was not statistically significant (p>0.05). Ramadan fasting induces weight loss, modifies gut microbiota, and improves blood lipid profile and FABP4 levels, suggesting the need for more extensive studies.