Background: Working in an underground tunnel environment is unavoidable in professions such as miners and tunnel workers, and there is a concern about the health of these workers. Few studies have addressed alterations in the intestinal microbiome of workers within that environment.Results: Fecal samples were collected from the workers before they entered the tunnel (baseline status, BS) and after they left the tunnel (exposed status, ES), respectively (a time period of 3 weeks between them). We analyzed 16S rRNA sequencing to show the changes in microbial composition and self-evaluation of mental health questionnaire was also performed. The results showed that Shannon and Simpson indices decreased significantly from BS to ES. A higher abundance was found in the phylumActinobacteria, classesActinobacteriaandDeltaproteobacteria, ordersBifidobacteriales,Coriobacteriales, andDesulfovibrionales, familiesBifidobacteriaceae,Peptostreptococcaceae,Coriobacteriaceae,Clostridiaceae_1,Desulfovibrionaceae, Pseudomonadaceae, and Microbacteriaceae, and generaBifidobacterium,Romboutsia,Clostridiumsensu stricto, andLeucobacterin ES, while BS showed greater levels of generaFaecalibacteriumandRoseburia. The self-evaluation showed that at least one-half of the tunnel workers experienced one or more symptoms of mental distress (inattention, sleeplessness, loss of appetite, headache or dizziness, irritability) after working in the underground tunnel environment.Conclusions: Collectively, the underground tunnel environment led to alterations in the intestinal microbiome, which might be relevant to symptoms of mental distress in underground-tunnel workers.