The largest family of toxin-antitoxin (TA) modules are encoded by the vapBC operons, but their roles in bacterial physiology remain enigmatic. Microarray analysis in Mycobacterium smegmatis overexpressing VapC/VapBC revealed a high percentage of downregulated genes with annotated roles in carbon transport and metabolism suggesting VapC was targeting specific metabolic mRNA transcripts. To validate this hypothesis, purified VapC was used to identify the RNA cleavage site in vitro. VapC had ribonuclease activity that was sequence-specific, cleaving single-stranded RNA substrates at AUAU and AUAA in vitro and in vivo (viz. MSMEG_2121-2124). A bioinformatic analysis of these regions suggested that an RNA hairpin 3' of the AUA(U/A) motif is also required for efficient cleavage. VapC-mediated regulation in vivo was demonstrated by showing that MSMEG_2124 (dhaF) and MSMEG_2121 (dhaM) were upregulated in a ?vapBC mutant growing on glycerol. The ?vapBC mutant had a specific rate of glycerol consumption that was 2.4-fold faster than the wild-type during exponential growth. This increased rate of glycerol consumption was not used for generating bacterial biomass suggesting that metabolism by the ?vapBC mutant was uncoupled from growth. These data suggest a model in which VapC regulates the rate of glycerol utilization to match the anabolic demands of the cell allowing for fine-tuning of the catabolic rate at a post-transcriptional level.