Anthropogenic nitrogen (N) enrichment is a concern worldwide, as it affects almost every aspect of ecosystem function and composition, particularly soil microbial communities that control soil organic matter (SOC) and nutrient turnover. Although many long-term nitrogen addition experiments have been conducted, there are still no conclusions about how microorganisms respond to nitrogen deposition, especially in mid-subtropical natural forests. Assays for soil microbial biomass and enzyme activity have become a common methodology for studying soil function in response to global environmental changes and disturbances. Between 2012 and 2015, we stimulated nitrogen deposition in a Castanopsis carlesii natural forest in Sanming Fujian Province in subtropical China. Soils were collected from four control plots (CT), four low-nitrogen addition plots (LN), and four high-nitrogen addition plots (HN). We studied the physicochemical properties of the soil; soil microbial C, N, and P biomasses; and potential activity of six enzymes in response to 3 years of nitrogen deposition. The results showed that nitrogen deposition had little impact on SOC and total nitrogen content. The HN treatment significantly decreased the pH and acidified the soil. Cellulose hydrolysis enzymes (β-glucosidase (βG) and cellobiohydrolase (CBH)) and lignin oxidases enzymes (phenol oxidase (PHO) and peroxidase (PEO)) showed a significant response to the LN treatment in the A horizon. Similarly, the LN treatment promoted microbial biomass carbon (MBC) and nitrogen (MBN) in the A horizon. Redundancy analysis (RDA) showed that dissolved organic carbon (DOC) greatly accounted for the variation in soil enzyme activities in the A horizon. However, it did not reveal any significant differences in cellulose hydrolysis and lignin oxidases in the B horizon. Moreover, nitrogen deposition promoted acid phosphatase activities in the two soil horizons. In summary, the LN treatment accelerated soil carbon turnover, promoting soil carbon mineralization. This may provide a theoretical basis for assessing whether mid-subtropical natural forest soil will become a carbon sink or source in an N-deposition background.