High-altitude (HA) visitors like pilgrims, trackers, scientists and military personnel face a group of nonspecific gastrointestinal (GI) symptoms during acclimatization to hypobaric hypoxia. In order to investigate the alteration of indigenous gastrointestinal microbiota in the development of such GI symptoms, an experiment was conducted for the enumeration of dominant cultivable faecal microbiota of 15 soldiers at base level (Delhi) and during their 15-day acclimatization at 3,505 m HA (Leh). At HA, faecal microbiota analysis revealed that total aerobes decreased significantly with increase of total and facultative anaerobes. The strict anaerobes like Bifidobacterium sp., Bacteroidetes sp. and Lactobacillus sp. exhibited positive growth direction index (GDI) like other predominant obligate anaerobes Clostridium perfringens and Peptostreptococcus sp. Different enzymes like amylase, proteinase and polyphenol hydrolase produced by different bacterial populations showed positive GDI, whereas phosphatase producers exhibited negative GDI. The levels of microbe-originated enzymes like amylase, proteinase, alkaline phosphatase and β-glucuronidase were also elevated during HA acclimatization. In addition, in vitro gas production ability was enhanced with increase of faecal immunoglobulins IgA and IgG. We demonstrated that hypoxic environment at HA had the potential to alter the gut microbial composition and its activities that may cause GI dysfunctions.