Nitrate attenuation during river bank infiltration is the key process for reducing nitrogen pollution. Temperature is considered to be an important factor affecting nitrate attenuation. However, the magnitude and mechanism of its impact have not been clear for a long time. In this study, the effects of temperature and temperature gradient on the nitrate denitrification rate were investigated via static batch and dynamic soil column simulation experiments. The results showed that temperature had a significant effect on the denitrification rate. Temperature effects were first observed in denitrifying bacteria. At low temperatures, the microorganism diversity was low, resulting in a lower denitrification rate constant. The static experimental results showed that the denitrification rate at 19°C was approximately 2.4 times that at 10°C. The dynamic soil column experiment established an exponential positive correlation between the nitrate denitrification decay kinetic constant and temperature. The affinity of denitrifying enzymes for nitrate in the reaction substrate was ordered as follows: decreasing temperature gradient (30°C → 10°C) > zero temperature gradient (10°C) > increasing temperature gradient condition (0°C → 10°C). This study provides a theoretical basis for the biogeochemical processes underlying river bank infiltration, which will help aid in the development and utilization of groundwater resources.