Type 2 diabetes mellitus (T2DM) is closely associated with changes in the composition of the gut microbiota. To date, studies on the gut microbiota have focused on the genus-level composition and microbial gene sets, whereas changes in the microbiota after clinical treatment have remained largely elusive. In the present study, 16 subjects with T2DM were enrolled and treated long-term with a low-fat diet. Stool samples were collected at the initial diagnosis and after 1, 3 and 6 months of treatment, and named as group T0, T1, T2 and T3, respectively. Simultaneously, stool samples from 16 healthy individuals were collected as a control (group C). In addition, 16S ribosomal RNA sequencing was performed to detect differences in the microbiota between the groups. Following the low-fat diet treatment, the patients' fasting plasma glucose, plasma glucose 2 h after challenge, glycosylated haemoglobin A1c and body mass index (BMI) decreased significantly. The composition of the phylum in patients with type 2 diabetes mellitus was similar to that in healthy individuals. A total of 23 genera from four phyla, namely Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria, were determined to be different between group T0 and group C, while only 8 genera were different between group T3 and group C. Repeated analysis of variance suggested a complex change during the low-fat diet treatment. The butyrate-producing bacteria Anaerotruncus exhibited a slight increase, while Roseburia was significantly increased at the T1 stage but then gradually decreased at the later stage. In summary, a low-fat diet was effective for patients with T2DM in reducing blood glucose and the BMI, and, to a certain extent, improving the intestinal flora to reach a normal composition. The study was registered in the Chinese Clinical Trial Registry (ChiCTR; registration no. ChiCTR1900028663).