Background and Aim: Simiao Yong'an decoction (SYD) is a classic traditional Chinese medicine (TCM) prescription that has the effects of clearing heat, detoxifying, promoting blood circulation, and relieving pain. In this study, we investigated the effect of SYD on the diversity of intestinal microbiota after fermentation by Bacillus subtilis. Materials and Methods: SYD was fermented using B. subtilis. Female Sprague-Dawley rats were randomly divided into the following four groups with six rats in each group: Negative sample group (NS), water exaction non-fermentation group (WE), B. subtilis group (BS), and fermentation liquid group (FL). All rats were orally administered for 14 days. High-throughput Illumina sequencing was used to analyze 16S rRNA expression in rat fecal samples. Results: A total of 2782 operational taxonomical units (OTUs) were identified in this study, and 634 OTUs were shared among all samples. Bacteroidetes (28.17%-53.20%) and Firmicutes (48.35%-67.83%) were the most abundant phyla identified among the four groups. The abundance of Escherichia and Alistipes was lower in the FL group than in the NS group, whereas the abundance of Bifidobacteria and Lactobacillus was increased in the FL group (p < 0.05). The abundance of Bifidobacterium was significantly upregulated in the FL group compared with the WE and BS groups (p < 0.05). Conclusion: After fermentation, SYD had a significantly better effect than SYD or B. subtilis. SYD significantly promoted the growth of intestinal probiotics, inhibited the growth of pathogenic bacteria, and maintained the balance of intestinal microbiota in SD rats. This study provides new insights into the development and use of SYD. Keywords: Bacillus subtilis, fermentation, intestinal microbiota, probiotics, Simiao Yong'an Decoction.