Resistant starch (RS) has been reported to reduce body fat in obese mice. However, this effect has not been demonstrated in humans. In this study, we tested the effects of RS in 19 volunteers with normal body weights. A randomized, double-blinded and crossover design clinical trial was conducted. The study subjects were given either 40 g high amylose RS2 or energy-matched control starch with three identical diets per day throughout the study. The effect of RS was evaluated by monitoring body fat, glucose metabolism, gut hormones, gut microbiota, short-chain fatty acids (SCFAs) and metabolites. The visceral and subcutaneous fat areas were significantly reduced following RS intake. Acetate and early-phase insulin, C-peptide and glucagon-like peptide-1 (GLP-1) secretion were increased, and the low-density lipoprotein cholesterol (LDL-C) and blood urea nitrogen (BUN) levels were decreased after the RS intervention. Based on 16S rRNA sequencing, certain gut microbes were significantly decreased after RS supplementation, whereas the genus Ruminococcaceae_UCG-005 showed an increase in abundance. Other potential signatures of the RS intervention included Akkermansia, Ruminococcus_2, Victivallis, and Comamonas. Moreover, the baseline abundance of the genera Streptococcus, Ruminococcus_torques_group, Eubacterium_hallii_group, and Eubacterium_eligens_group was significantly associated with the hormonal and metabolic effects of RS. These observations suggest that a daily intake of 40 g of RS is effective in modulating body fat, SCFAs, early-phase insulin and GLP-1 secretion and the gut microbiota in normal-weight subjects.