A laboratory incubation experiment was conducted to examine the effects of 3,4-dimethylpyrazole phosphate (DMPP) on the transformation of urea-N and associated microbial communities in a low-fertility brown soil. The soil was supplied with urea at 180 kg N/ha and DMPP at 0, 1.8 or 18 kg/ha, while soil that was not supplied with urea and DMPP was treated as the control. Soil mineral nitrogen (NH₄⁺-N and NO₃^–-N) was measured at regular intervals, and temporal variations in the population sizes of nitrogen-cycling microbes were determined using real-time PCR. Compared to the urea-alone treatment, the additional application of DMPP maintained significantly higher (P < 0.05) NH₄⁺-N concentrations and lower NO₃^–-N (P < 0.05) concentrations in the soil and decreased the population sizes of ammonia oxidizing and denitrifying microbes but promoted the growth of nitrogen-fixing bacteria. The results suggest that the application of DMPP is beneficial in improving the availability of fertilizer-N for plant uptake. This study is the first comprehensive investigation to examine the effects of DMPP on the functional microorganisms that are important in nitrogen-cycling processes.