Objective To study the effect of drinking water at different pH on intestinal microbial diversity in SPF mice. Methods Ninety 21-day-old SPF mice were randomly divided into three groups:drinking acidified water (pH 3.0), neutral water (pH 7.0) and alkaline water (pH 9.0), respectively, for 3 months, 30 mice in each group. 10 mice were sacrificed at the end of 4, 8 and 12 weeks in each group. Bacteria, virus and parasites in serum were detected according to the national standard for SPF. Then, primer sequences were designed according to the conservativeness of bacteria 16S rRNA, and libraries were constructed. Subsequently, the ileocecal contents were subjected to high-throughput sequencing (Illumina). 10 G data were obtained and analyzed. Results The sequencing results revealed that the intestinal microbial abundance of the mice receiving pH 3.0 drinking water was moderate with a high stability. Conclusions Under the conditions of large-scale breeding, the mice receiving pH 3.0 drinking water can maintain the intestinal microbial diversity for a long time, and reach a stable platform. This study provides theoretical and technical support for the stable long-term maintenance of animal quality production. It is also a specific application of gastrointestinal macrogenomics in animal quality control.