Rhizodeposition represents a readily available C and energy source for soil microorganisms, that plays an important role in the regulation of C and nutrient cycling in ecosystems and exerts a strong influence on C sequestration. The dynamics of rice rhizo-C in soils and its allocation to microorganisms during rice growth, as well as the effects of nitrogen (N-NH4+) fertilization are poorly understood, particularly with respect to the initial uptake of rhizo-C by microorganisms and its utilization during the entire growth period. To assess these two processes, rice plants were grown in pots with or without N fertilization (0 and 225 kg N-NH4+ ha−1), and 13C incorporation into microbial groups was traced by phospholipid fatty acids (PLFAs) analysis within 6 h after 13CO2 pulse labeling. Labeling was performed at five growth stages: tillering, elongation, heading, filling, and maturation. 13C incorporated into soil microbial biomass C changed rapidly at the beginning of the study period, before elongation, but remained stable thereafter. 13C incorporation into rhizosphere and bulk soil microbial biomass was higher with than without N addition. This stimulation was likely due to the excessive increase in phytomass formation and root exudates after N fertilization and the increased assimilate C input into the soil. Structural equation modelling suggested that N fertilization strongly affected carbon transfer between rhizosphere and non-rhizosphere. Hence, N-NH4+ application may not only increase rhizo-C flow into microorganisms but it may also increase the effect of rhizosphere on bulk-soil microorganisms and subsequent processes related to soil C-cycling.