Acidification of drinking water to a pH between 2.5 and 3.0 is widely used to prevent the spread of bacterial diseases in animal colonies. Besides hydrochloric acid (HCl), sulfuric acid (H2SO4) is also used to acidify drinking water. Here we examined the effects of H2SO4-acidified drinking water (pH = 2.8) received from weaning (postnatal day 21) on the behavior and gut microflora of 129S6/SvEv mice, a mouse strain commonly used in transgenic studies. In contrast to HCl-acidified water, H2SO4-acidified water only temporarily impaired the pole-descending ability of mice (at 3 months of age), and did not change the performance in an accelerating rotarod test. As compared to 129S6/SvEv mice receiving non-acidified or HCl-acidified drinking water, the gut microbiota of 129S6/SvEv mice on H2SO4-acidified water displayed significant alterations at every taxonomic level especially at 6 months of age. Our results demonstrate that the effects of acidified drinking water on the behavior and gut microbiota of 129S6/SvEv mice depends on the acid used for acidification. To shed some light on how acidified drinking water affects the physiology of 129S6/SvEv mice, we analyzed the serum and fecal metabolomes and found remarkable, acidified water-induced alterations.