Sucralose is an artificial non-nutritive sweetener used in foods aimed to reduce sugar and energy intake. While thought to be inert, the impact of sucralose on metabolic control has shown to be the opposite. The gut microbiome has emerged as a factor shaping metabolic responses after sweetener consumption. We examined the short-term effect of sucralose consumption on glucose homeostasis and gut microbiome of healthy male volunteers. We performed a randomized, double-blind study in 34 subjects divided into two groups, one that was administered sucralose capsules (780 mg/day for seven days; n=17), and a control group receiving placebo (n=17). Before and after the intervention, glycemic and insulinemic responses were assessed with a standard oral glucose load (75 g). Insulin resistance was determined using HOMA-IR and Matsuda indexes. The gut microbiome was evaluated before and after the intervention by 16S rRNA sequencing. During the study, body weight remained constant in both groups. Glycemic control and insulin resistance were not affected during the 7-day period. At the phylum level, gut microbiome was not modified in any group. We classified subjects according to their change in insulinemia after the intervention, to compare the microbiome of responders and non-responders. Independent of consuming sucralose or placebo, individuals with a higher insulinemic response after the intervention had lower Bacteroidetes and higher Firmicutes abundances. In conclusion, consumption of high doses of sucralose for 7 days does not alter glycemic control, insulin resistance, or gut microbiome in healthy individuals. However, it highlights the need to address individual responses to sucralose.