Increasing evidence indicates that molecular hydrogen-dissolved alkaline electrolyzed water (AEW) has various physiological activities such as antioxidative activity. Gut microbiota are deeply associated with our health through a symbiotic relationship. Recent reports have described that most gastrointestinal microbial species encode the genetic capacity to metabolize molecular hydrogen, meaning that molecular hydrogen might affect the gut microbial composition. Nevertheless, AEW effects on gut microbiota remain unknown. This study investigated AEW effects on the intestinal environment in mice, including microbial composition and short-chain fatty acid contents. After mice were administered AEW for 4 weeks, 16S rRNA gene sequencing analyses revealed their fecal microbiota profiles. Organic acid concentrations in cecal contents were measured using an HPLC system. Compared to the control group, AEW administration mice had significantly lower serum low-density lipoprotein cholesterol level and alanine aminotransferase activity. Organic acid concentrations of propionic, isobutyric, and isovaleric acids were higher in AEW-administered mice. Results of 16S rRNA gene sequencing analyses showed that the relative abundances of 20 taxa differed significantly in AEW-administered mice. Although the definitive role of gut microbes of AEW-administered mice remains unknown, our data demonstrate the possibility that AEW administration affects the gut microbial composition and that it has beneficial health effects in terms of cholesterol metabolism and liver protection. Higashimura Yasuki 1 Department of Food Science, Ishikawa Prefectural University, Nonoichi, Ishikawa; Molecular Gastroenterology and Hepatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Kyoto Baba Yasunori 2 Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa Inoue Ryo 3 Laboratory of Animal Science, Department of Agricultural and Life Sciences, Kyoto Prefectural University, Kyoto Takagi Tomohisa 4 Molecular Gastroenterology and Hepatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Kyoto Uchiyama Kazuhiko 5 Molecular Gastroenterology and Hepatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Kyoto Mizushima Katsura 6 Molecular Gastroenterology and Hepatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Kyoto Hirai Yasuko 7 Molecular Gastroenterology and Hepatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Kyoto Ushiroda Chihiro 8 Molecular Gastroenterology and Hepatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Kyoto Tanaka Yoshinori 9 Appliances Company, Panasonic Corporation, Shiga Naito Yuji 10 Molecular Gastroenterology and Hepatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Kyoto Bajzer M, Seeley RJ. 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