Abstract: In this study, the effects of intercropping with alfalfa and nitrogen application on the functional diversity of soil microbial community in mulberry rhizosphere were examined by Biolog-Ecoplate^TM technique, and principal component and canonical analyses. Compared to monoculture with no nitrogen (N) addition, monoculture with N application and intercropping with alfalfa remarkably reduced soil pH and significantly increased the contents of soil organic matter, soil available N, soil water content, and activities of peroxidase and urease. Monoculture with N application and intercropping with alfalfa (with or without N application) increased the AWCD values, diversity index, and the carbon source utilization ratios of soil microbes. Higher increments of these parameters were detected in the treatment of intercropping plus N application. The results of principal component analysis showed that N application and intercropping changed the capacity of the rhizosphere microbial community for utilizing carbon sources. The utilization of carbon sources highly related to the principal components by the rhizosphere microbial communities was similar in the treatments of monoculture with N application and intercropping without N application. The utilization of itaconic acid and D-glucamaminic acid in the latter was more than 4% and was significantly higher than that in the former. The results from redundancy analysis showed that the soil microbial diversity in mulberry rhizosphere of the treatment of monoculture without N application was positively correlated with polyphenol oxidase activity and negatively correlated with soil water content, whereas that of monoculture with N application and intercropping without N application was significantly positively correlated with soil pH and soil water content and negatively correlated with soil N avalaibility. The diversity of the microbes in the rhizosphere soil of mulberries under the treatment of intercropping with N application showed positive correlation with soil N availability and was significantly negatively correlated with soil pH.