To estimate the effect of long-term fertilization on metabolically active bacterial communities in a rice field, RNA was extracted from endosphere (rice root), rhizosphere, and bulk soil that had been subjected to different fertilization regimes for 59 years, and the 16S rRNAs were analyzed using the pyrosequencing method. The richness and diversity of metabolically active bacteria were higher in bulk soil than in the endosphere and rhizosphere, and showed no significant difference between non-fertilized and fertilized plots. Weighted UniFrac analysis showed that each compartment had characteristic bacterial communities and that the effect of long-term fertilization on the structure of bacterial community was more pronounced in bulk soil than in the endosphere and rhizosphere. The 16S rRNAs affiliated with Alphaproteobacteria and Firmicutes were more abundant in the endosphere than in bulk soil while those affiliated with Chloroflexi and Acidobacteria were more abundant in bulk soil than in the endosphere. Several dominant operational taxonomic units (clustered at a 97% similarity cut-off) showed different frequencies between non-fertilized and fertilized plots, suggesting that the fertilization affected their activities in the rice field.