Quorum sensing (QS) is a process of cell-cell communication that enables bacteria to transition between individual and collective lifestyles. QS controls virulence in Vibrio cholerae, the causative agent of the disease cholera. Differential RNA-sequencing (dRNA-seq) analyses of wild-type V. cholerae and a locked low cell density QS-mutant strain identified a total of 7641 transcriptional start sites (TSS) with ~40% initiated in the antisense direction. Genome-wide TSS mapping combined with phylogenetic comparisons enabled re-annotation of 129 genes from the NCBI database. 107 of the transcripts we identified do not appear to encode proteins suggesting they could specify non-coding RNAs. We focused on one such transcript that we name VqmR. vqmR is located upstream of the vqmA gene that encodes a DNA-binding transcription factor. Mutagenesis and microarray analyses demonstrate that VqmA activates vqmR transcription that vqmR encodes a regulatory RNA, and VqmR directly controls at least eight mRNA targets including the rtxBA toxin genes and the vpsT transcriptional regulator of biofilm production. We show that VqmR inhibits biofilm formation through repression of vpsT. Together, these data provide the first global annotation of the V. cholerae transcription landscape and they highlight the importance of post-transcriptional regulation for collective behaviors in this human pathogen. Overall design: Global mapping TSS in V. cholerae