With simulated pot culture in greenhouse, the ammonia volatilization from rice shoot and its affecting factors after flowering was studied under different N fertilization levels. The results showed that the daily and total amount of volatilized ammonia increased with increasing N application rate, with a certain variance among different N levels observed. At flowering and maturing stages; the ammonia volatilization from rice shoot (y) had significant negative correlations with functional leaf GS activity (x1) and positive correlations with functional leaf apoplastic NH4+ concentration (x2), with y = -0.37846x1 + 0.41821x2 + 0.04925 (R2 = 0.9471, n = 16), and negative correlations with N harvest index (x1) and physiological N use efficiency (x2), following y = -0.02117x1 + 0.75186 (R2 = 0.842, n = 8) and y = -1.10386x2 + 35.52676 (R2 = 0.8489, n = 8), respectively. It was suggested that the increasing ammonia volatilization from rice shoot under high N fertilization level was correlated with the decrease of N use efficiency by rice.