The application of urea in agricultural soil significantly boosts nitrous oxide (N2O) emissions. However, the reason for nitrite accumulation, the period of nitrite-oxidizing bacteria (NOB) suppression, and the main NOB species for nitrite removal behind urea fertilization have not been thoroughly investigated. In this study, four laboratory microcosm experiments were conducted to simulate urea fertilization in agricultural soils. We found that within 36 h of urea application, nitrite oxidation lagged behind ammonia oxidation, leading to nitrite accumulation and increased N2O emissions. However, after 36 h, NOB activity recovered and then removed nitrite, leading to reduced N2O emissions. Urea use resulted in an N2O emission rate tenfold higher than ammonium. During incubation, Nitrobacter-affiliated NOB growth decreased initially but increased later with urea use, while Nitrospira-affiliated NOB appeared unaffected. Chlorate suppression of NOB lasted longer, increasing N2O emissions. Urease inhibitors effectively reduced N2O emissions by slowing urea hydrolysis and limiting free ammonia production, preventing short-term NOB suppression. In summary, short-term NOB suppression during urea hydrolysis played a crucial role in increasing N2O emissions from agricultural soils. These findings revealed the reasons behind the surge in N2O emissions caused by extensive urea application and provided guidance for reducing N2O emissions in agricultural production processes.