Objective: In this study, we investigated the composition of the intestinal microflora associated with Yang-deficiency and the function-specific members of this microflora. Method: Subjects with normal and Yang-deficient constitutions were recruited in Beijing, with 30 subjects in each group. Illumina high-throughput sequencing was used to sequence the DNA of their fecal bacteria in the 16S rRNA V3–V4 region. The species abundance and distribution of the intestinal microflora in each specimen were determined with a statistical analysis of these sequences. We identified an underlying taxonomic trend with nonparametric PCoA and other statistical techniques. Results: (1) Subjects with Yang-deficiency displayed more uneven abundances of the taxa in their intestinal microflora than the subjects with normal Yang; (2) several differences were observed in the proportions of certain bacterial groups between the Yang-deficient and control groups; and (3) the following bacteria differed significantly between the two groups: (i) Brevundimonas, Leuconostoc, Turicibacter, Defluviitaleaceae incertae sedis, Rothia, and Butyricimonas were slightly higher in the Yang-deficient subjects, whereas (ii) Megasphaera, Ruminococcus, and Lachnospira were higher in the normal subjects than in the Yang-deficient subjects. Conclusions: The composition of the intestinal microflora is altered in Yang-deficient subjects.