High habitual consumption of non-nutritive sweeteners (NNS) is linked to increased incident type 2 diabetes, with emerging clinical evidence that effects on gut microbiota may, in part, drive this risk. However, the precise contribution of the effects of NNS on gut microbiota to host glycemic responses remains unclear. Ten-week-old male C57BL/6 mice (N = 10 per group) were randomized to drinking water with or without combined NNS (sucralose 1.5 mg/mL plus acesulfame-K 2.5 mg/mL) and with or without antibiotics to deplete gut microbiota (ABX, 1 mg/mL ampicillin and neomycin) over two weeks. Oral glucose tolerance tests (OGTT, 2 g/kg) were conducted on days -1 and 12. On day 14, mice underwent a jejunal infusion of glucose (300 mg) with 3-O-methyl glucose (30 mg, 3-OMG, a marker of glucose absorption) in 1.5 mL for 30 min, followed by blood collection and bioassays. Data were analyzed using ANOVA with NNS and ABX as factors. Jejunal glucose absorption was augmented in NNS+ mice relative to NNS- (31%; 3-OMG T30; ≤ 0.05) independent of ABX. ABX attenuated OGTT responses independent of NNS supplementation (-35%; incremental AUC, ≤ 0.001). NNS+ ABX+ mice had augmented GLP-1 responses to intrajejunal glucose relative to other groups (69-108%, < 0.05). These findings demonstrate that sub-acute NNS supplementation augments glucose absorption independent of gut microbiota in mice but does not disrupt glycemic responses. Antibiotic depletion of gut microbiota markedly increased glucose tolerance in mice, which may involve the actions of GLP-1.