Mulberry intercropped with alfalfa is a popular agroforestry system in China, which can produce high forage yields with high protein. To investigate the advantages of intercropping as well as the responses of mulberry-alfalfa intercropping systems to nitrogen application, we studied changes in the soil microbial communities and physiochemical properties in the rhizosphere of intercropped mulberry and alfalfa under nitrogen application. Nitrogen application increased available nitrogen contents (AN) and activities of urease (SUR) in rhizosphere soil of mulberry and alfalfa, but reduced soil organic canmatter (OM), irrespective of the cropping system (monoculture or intercropping). Nitrogen application increased soil pH in the rhizosphere of mulberry and alfalfa in monoculture, but reduced soil pH in the intercropped system. Nitrogen application and intercropping enhanced the soil water content (SWC) in mulberry rhizosphere soil and decreased SWC in alfalfa rhizosphere soil. We observed significant differences in the microbial communities inhabiting mulberry and alfalfa rhizosphere soils, indicating that mulberry and alfalfa are strategically complementary in terms of carbon sources. The Shannon-Weaver (H’), Simpson index (D), and McIntosh diversity (U) values were higher in rhizosphere soil of intercropped mulberry compared with mulberry in monoculture without nitrogen supply. There were no significant differences between mulberry in monoculture with nitrogen application (MNE) and mulberry intercropped alfalfa without nitrogen (M0). The results of the principal components analysis showed that MNE and M0 clustered in the scattered plots. We found no significant differences for H’ and D between monoculture and intercropping alfalfa systems, irrespective of the nitrogen application. Nitrogen application, intercropping, and intercropping with nitrogen increased the numbers of carbon sources, and relative use rates exceeded 4%. Nitrogen application and intercropping reduced the numbers of available carbon sources in alfalfa. Redundancy discriminatory analysis results suggest that pH and SWC were positively related with mulberry treatments and negatively with the alfalfa treatments.