An increasing body of evidence suggests that gut microbiota is involved in atopic dermatitis (AD). We aimed to use high-throughput sequencing to characterize the differences in the composition of the gut microbiota between healthy controls and patients with AD. Fecal samples from 93 volunteers were analyzed using 16S rRNA sequencing, including 44 patients with AD and 49 healthy control subjects, aged 6-22 years. Our data show that the operational taxonomic unit composition in patients with AD had greater component similarity than the healthy controls. Patients with AD had a lower alpha diversity than healthy control subjects. The relative abundance of Porphyromonadaceae, Blautia, Parabacteroides, Bacteroides ovatus, Bacteroides uniformis and Prevotella stercorea was significantly higher (P < 0.05) in patients with AD than healthy control subjects. Clostridium and P. stercorea were higher (P < 0.05) in healthy control subjects compared with patients with AD. The results of linear discriminant analysis effect size show that Bacteroidaceae and Porphyromonadaceae can act as possible biomarkers associated with diagnosis of AD. However, this needs further experimental verification. Taken together, these results demonstrate the changes in microbiota composition in AD compared with a healthy control group, opening the way to future diagnosis or intervention studies. © 2020 Japanese Dermatological Association.