Vibrio cholerae, the causative agent of cholera, is a facultative human pathogen with intestinal and aquatic life cycles. The capacity of V. cholerae to recognize and respond to fluctuating parameters in its environment is critical to its survival. In many microorganisms, the second messenger, 3′,5′-cyclic diguanylic acid (c-di-GMP), is believed to be important for integrating environmental stimuli that affect cell physiology. Sequence analysis of the V. cholerae genome has revealed an abundance of genes encoding proteins with either GGDEF domains, EAL domains, or both, which are predicted to modulate cellular c-di-GMP concentrations. To elucidate the cellular processes controlled by c-di-GMP, whole-genome transcriptome responses of the El Tor and classical V. cholerae biotypes to increased c-di-GMP concentrations were determined. The results suggest that V. cholerae responds to an elevated level of c-di-GMP by increasing the transcription of the vps, eps, and msh genes and decreasing that of flagellar genes. The functions of other c-di-GMP-regulated genes in V. cholerae are yet to be identified.