: Artificial non-nutritive sweeteners (NNSs), such as sucralose, have been associated with gut microbiota (GM) alterations. However, the impact of rebaudioside A (reb A), a natural NNS, on GM has received limited scrutiny. Objective: The objective of this study was to examine the response of GM composition to sucralose and reb A in rats under two dietary conditions. : Male Wistar rats (150-200 g) fed with a normal diet (ND) or a high-fat diet (HFD) were randomly assigned to receive sucralose (SCL), reb A (REB), glucose (GLU, control), or sucrose (SUC). The NNS interventions were administered in water at doses equivalent to the acceptable daily intake (ADI). After eight weeks, the GM composition in fecal samples was analyzed through 16S ribosomal RNA gene sequencing. : The NNSs did not modify the diversity, structure, phylum-level composition, or Firmicutes/Bacteroidetes (F/B) ratio of the GM in rats under ND or HFD. However, REB with HFD decreased and increased abundance at the class level. SCL and REB in rats receiving ND reduced the genera and . : Our study suggests that when sucralose or reb A is consumed at recommended doses, there is no alteration in the diversity or the composition of the GM at the phylum level. The clinical relevance of these findings lies in the potential modifications of the GM at specific taxonomic levels by the consumption of these NNSs. Further research involving humans and including a broader range of microbial analyses is warranted.