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Pathogens adapt to the host environment by altering their patterns of gene expression. Microarray-based and genetic techniques used to characterize bacterial gene expression during infection are limited in their ability to comprehensively and simultaneously monitor genome-wide transcription. We used massively parallel cDNA sequencing (RNA-seq) techniques to quantitatively catalog the transcriptome of the cholera pathogen, Vibrio cholerae, derived from two animal models of infection. Transcripts elevated in infected rabbits and mice relative to laboratory media derive from the major known V. cholerae virulence factors and also from genes and small RNAs not previously linked to virulence. The RNA-seq data was coupled with metabolite analysis of cecal fluid from infected rabbits to yield insights into the host environment encountered by the pathogen and the mechanisms controlling pathogen gene expression. RNA-seq-based transcriptome analysis of pathogens during infection produces a robust, sensitive, and accessible data set for evaluation of regulatory responses driving pathogenesis.