Studies in rodent models have shown that alterations in drinking water pH affect both the composition of the gut microbiota and host glucose regulation. To explore a potential impact of electrochemically reduced alkaline (pH ≈ 9) versus neutral (pH ≈ 7) drinking water (2 L/day) on human intestinal microbiota and host glucose metabolism we conducted a randomized, non-blinded, cross-over study (two 2-week intervention periods, separated by a 3-week wash-out) in 29 healthy, non-smoking Danish men, aged 18 to 35 years, with a body mass index between 20.0 to 27.0 kg m-2. Volunteers were ineligible if they had previously had abdominal surgery, had not been weight stabile for at least two months, had received antibiotic treatment within 2 months, or had a habitual consumption of caloric or artificially sweetened beverages in excess of 1 L/week or an average intake of alcohol in excess of 7 units/week. Microbial DNA was extracted from faecal samples collected at four time points, before and after each intervention, and subjected to 16S rRNA gene amplicon sequencing (Illumina MiSeq, V4 region). Glycaemic regulation was evaluated by means of an oral glucose tolerance test.No differential effect of alkaline versus neutral drinking water was observed for the primary outcome, overall gut microbiota diversity as represented by Shannon's index. Similarly, neither a differential effect on microbiota richness or community structure was observed. Nor did we observe a differential effect on the abundance of individual operational taxonomic units (OTUs) or genera. However, analyses of within period effects revealed a significant (false discovery rate ≤5%) increase in the relative abundance of 9 OTUs assigned to order Clostridiales, family Ruminococcaceae, genus Bacteroides, and species Prevotella copri, indicating a potential effect of quantitative or qualitative changes in habitual drinking habits. An increase in the concentration of plasma glucose at 30 minutes and the incremental area under the curve of plasma glucose from 0 30 and 0 120 minutes, respectively, was observed when comparing the alkaline to the neutral intervention. However, results did not withstand correction for multiplicity. In contrast to what has been reported in rodents, a change in drinking water pH had no impact on the composition of the gut microbiota or glucose regulation in young male adults. The study is registered at www.clinicaltrials.gov (NCT02917616).