The present study investigated long-term treatment performance and nitrogen transformation mechanisms in tidal flow constructed wetlands (TFCWs) under 4, 8, and 12 °C temperature regimes. High and stable ammonium (NH4+-N) removal efficiency (93–96%) was achieved in our TFCWs, whereas nitrate (NO3–-N) was accumulated at different levels under different temperatures. Quantitative response relationships showed anammox/amoA, (narG+napA)/amoA, and (narG+napA)/bacteria were the respective key functional gene groups determining 4, 8, and 12 °C NO3–-N reduction. Pathway analysis revealed the contribution of these functional gene groups along a depth gradient. In addition, denitrification process increased, while anammox process decreased consistent with a rise in temperature from 4 to 12 °C. Furthermore, cold temperatures exhibited different effects on anammox and denitrification and their long-term acclimatization capacities changed with temperature.