Environmental factors can influence the composition of gut microbiota, but understanding the combined effect of lifestyle factors on adult gut microbiota is limited. Here, we investigated whether changes in the modifiable lifestyle factors, such as cigarette smoking, alcohol consumption, sleep duration, physical exercise, and body mass index affected the gut microbiota of Korean navy trainees. The navy trainees were instructed to stop smoking and alcohol consumption and follow a sleep schedule and physical exercise regime for eight weeks. For comparison, healthy Korean civilians, who had no significant change in lifestyles for eight weeks were included in this study. A total of 208 fecal samples were collected from navy trainees (n = 66) and civilians (n = 38) at baseline and week eight. Gut flora was assessed by sequencing the highly variable region of the 16S rRNA gene. The α-and β -diversity of gut flora of both the test and control groups were not significantly changed after eight weeks. However, there was a significant difference among individuals. Smoking had a significant impact in altering α-diversity. Our study showed that a healthy lifestyle, particularly cessation of smoking, even in short periods, can affect the gut microbiome by enhancing the abundance of beneficial taxa and reducing that of harmful taxa.