Aiming for total nitrogen (TN) pollution control in the urbanized stream, this study proposed and verified a strategy of cultivating and acclimating sulfur-based autotrophic denitrifiers by using river-bottom sediments as seed sludge, and investigated temperature effects on sulfur-based autotrophic denitrification (SAD). With thiosulfate as an electron donor, seven SAD batch reactors were operated and studied at both 15 °C and 30 °C, to compare reactor performance and their microbial community analysis results. In the first batch, three parallel reactors (A1, A2, and A3) were operated at 30 °C for 30 days. The dynamic analysis showed that sequentially decreasing temperature to 20, 15, and 10 °C had significant adverse effects on nitrate-loading rates. In the second batch, two groups of parallel reactors were operated at 30 °C (B1 and B2) and 15 °C (C1 and C2) for 45 days. High TN removal efficiencies (>95%) were achieved in all four reactors, with comparable nitrate loading rates and less nitrite accumulation at 15 °C. High-throughput sequencing revealed that genus Thiobacillus was predominant (66.3–90.0%) in all seven reactors. However, at the operational taxonomic unit level, microbial communities at 15 °C and 30 °C were significantly different, indicating that dissimilar strains were cultivated. Our findings suggested that deliberately cultivating cold-adapted denitrifiers helps SAD to achieve high TN removal at psychrophilic temperatures and thus, is important for future applications in practical TN pollution control in urbanized streams.