The rapid expansion of Solidago canadensis and climate warming in southeastern China may interactively affect soil net nitrogen (N) mineralization, which may lead to plant invasion. A greenhouse simulated experiment was conducted with invasion, warming, and their interaction to investigate these changes’ effects on soil net N mineralization in an ecological system. Our results indicated that the average rate of net mineralization, nitrification, and ammonification decreased with invasion, warming, and their interaction. The enzyme activities and pH showed more sensitivity in warming than invasion, and have a similar decreased trend with net N mineralization response to environmental changes, except sucrase. At the same time, enzyme and pH may play a key role in the process of net N mineralization from Pearson’s correlation and redundancy analysis, especially for sucrase and urease. In addition, the lower produced of litter biomass by plants growing in pots was also an important reason for the decrease of net N mineralization rate. These results indicated that the significant decrease in substrate quality (N availability) by the increase in invasion and warming may cause the deterioration of species production in soil, which will have important consequences for soil ecology, N-cycles, and plant invasion.