Episodic inputs of labile carbon (C) to soil can rapidly stimulate nitrogen (N) immobilization by soil microorganisms. However, the transcriptional patterns that underlie this process remain unclear. In order to better understand the regulation of N cycling in soil microbial communities, we conducted a 48-h laboratory incubation with agricultural soil where we stimulated the uptake of inorganic N by amending the soil with glucose. We analyzed the metagenome and metatranscriptome of the microbial communities at four time points that corresponded with changes in N availability. The relative abundances of genes remained largely unchanged throughout the incubation. In contrast, glucose addition rapidly increased the transcription of genes encoding ammonium and nitrate transporters, enzymes responsible for N assimilation into biomass, and genes associated with the N regulatory network. This upregulation coincided with an increase in transcripts associated with glucose breakdown and oxoglutarate production, demonstrating a connection between C and N metabolism. When concentrations of ammonium were low, we observed a transient upregulation of genes associated with the nitrogen-fixing enzyme nitrogenase. Transcripts for nitrification and denitrification were downregulated throughout the incubation, suggesting that dissimilatory transformations of N may be suppressed in response to labile C inputs in these soils. These results demonstrate that soil microbial communities can respond rapidly to changes in C availability by drastically altering the transcription of N cycling genes.