To address the impact of low-temperature pH on the microbial community structure of river sediment in urban rivers during winter, the sediment of the Pi River was sampled to investigate. Four experimental samples were designed by varying the pH to 5.0, 7.0, and 8.5 based on the original sample (pH = 5.5) and the characteristics of the microbial community were analyzed using the high-throughput sequencing. The results showed that the samples contained 23 phyla, 68 classes, and 100 genera of bacteria, of which Sphingomonales and Burkholderiales were the dominant genera. The highest abundance of Sphingomonales was 20%, whereas the highest abundance of Burkholderiales was up to 15% in the acid samples. The abundance of Proteobacteria was the highest in the original and alkaline samples, followed by Bacteroides and the number of other genera in alkaline and neutral samples is more than that in acidic samples. In addition, the abundance of Nitrosomonadales was more than 5% in the original, alkaline, and neutral samples and less than 1% in acidic conditions. The microbial community of the acid sample was quite different from the other three samples, with less richness and uniformity, which indicated that the acid pH had a great impact on the microbial community and the denitrifying bacteria were less active at the low-temperature condition in winter. This study provides a reference for the microbial community structure of the Pi River and a scientific basis for effectively addressing urban pollution in river management.