AimsThe microbe phoD gene, which encodes alkaline phosphatase (ALP), is crucial for mineralizing organic phosphorus (Po) to bioavailable P. However, the effect of long-term nitrogen (N) addition on the phoD-harbouring microbial-mediated P cycle and its mechanisms are poorly understood.MethodsWe performed an 8-year N application experiment on alfalfa in the Loess Plateau of China. Moreover, the soil P forms, ALP activity, and phoD-harbouring microbial community characters under different N addition rates (0, 50, 100, and 150 N ha-1 yr-1) were determined.ResultsLong-term N addition significantly reduced soil phoD gene abundance, pH, AP, labile P, and ALP activity but significantly increased the percentages of NaOH-Po and residual P in total P. N application significantly changed the community composition of phoD microbes. Furthermore, we found that N application significantly reduced the dispersal limitation of stochastic processes, N50, N100, and N150 treatments decreased by 53.17%, 89.95%, and 85.82%, respectively, compared with the N0 treatment. Network analysis indicated that the microbial network in the high-N group showed higher links (354), average degree (7.08), closeness centrality (0.301), and neighborhood connectivity (7.644) compared to those of the low-N group microbial network (6.52, 0.292, and 7.057, respectively). According to structure equation models (SEM), the key factors determining ALP were N addition rates, phoD gene abundance, and soil pH.ConclusionsOur study indicated that N application can promote microbial competition for P and reduce soil P availability via phoD-harbouring microorganisms in the alfalfa continuous cropping system.