A novel hypothermic strain, Pseudomonas fragi EH-H1, was found to effectively perform heterotrophic nitrification and aerobic denitrification at 15 °C. This strain could consume 100 %, 100 % and 99.95 % of ammonium (54.90 mg∙L⁻¹), nitrate (56.12 mg∙L⁻¹) and nitrite (54.15 mg∙L⁻¹), accompanied by peak removal rates of 5.51, 3.63 and 3.14 mg/L/h, respectively. The ammonium was removed preferentially during simultaneous nitrification and denitrification. Notably, the elimination rate of the toxic nitrite nitrogen remained approximately 3.14 mg/L/h, whether supplemented with ammonium or not. Stepwise inhibition experiments revealed that the key enzymes of ammonia monooxygenase (AMO) and nitrite oxidoreductase (NiR) for nitrification and denitrification coexisted in strain EH-H1. AMO, nitrate reductase and NiR were successfully expressed and detected at 0.637, 0.239 and 0.018 U/mg proteins, respectively. Overall, strain EH-H1 had an outstanding ability to remove nitrogen at low temperatures and could provide guidance for cryogenic wastewater treatment.