Alternate wetting and drying (AWD) irrigation influences soil nutrient cycling and the functioning of soil microorganisms. However, the effects of AWD on organic nitrogen (ON) partitioning in rice-microbe ecosystems and soil microbial communities are poorly understood. A root-box microcosm experiment with two rice varieties two irrigation regimes (conventional flood irrigation (CF); AWD) and three N application levels (zero N, N0; medium N, N1; high N, N2) was performed based on the 13C,15N-labelled glycine and 13C-phospholipid fatty acids (PLFA) techniques. Compared to CF, AWD increased soil dissolved oxygen, microbial growth and the enzymes related to N transformation, thus enhancing rice growth and the N utilization index (NUI). Approximately 4.9–10.3% and 7.7–13.6% of the exogenous glycine was directly utilized by Nip and YD6 seedlings, respectively, and its ratio increased with increasing N levels, whereas 4.4–11.2% and 4.6–10.3% were incorporated into soil microbes. It seems that rice has an appreciable capacity to utilize organic N despite fierce competition with soil microbes. The 13C:15N ratio showed that 12.5–37.5% and 11.0– 41.0% of the added glycine was taken up intact by soil microorganisms in the rhizospheres of Nip and YD6. At the N1 and N levels, AWD increased rice 15N-glycine uptake but decreased microbial 15N-glycine uptake. Rice intact glycine uptake and soil inorganic N contents were positively correlated with rice biomass and NUI, indicating that the enhanced inorganic N under AWD is beneficial for soil ON availability and rice growth.