Borrelia burgdorferi (Bb), the causative agent of Lyme disease, must adapt to vastly different environments as the bacterium cycles between the tick vector and a vertebrate host. During a bloodmeal, Bb migrates from the tick midgut to the salivary glands and changes its gene expression, priming Bb for vertebrate infection. These tick-specific events are dependent on key transcriptional regulators; however, the full range of transcriptional changes that occur inside of the tick over the course of several days during transmission are technically difficult to capture. We developed an experimental approach to enrich Bb cells from Ixodes ticks during a transmitting bloodmeal to longitudinally define their global transcriptomic landscape. We identified 192 genes that change expression at least twofold over the course of the tick bloodmeal, which were predominantly located on plasmids of the Bb genome. The majority of upregulated genes encode proteins found at the cell envelope or proteins of unknown function, including 45 upregulated outer surface lipoproteins embedded in the unusual protein-rich coat of Bb. The ex vivo Bb transcriptomes provide an important roadmap for investigating key determinants of Bb priming and transmission during the tick stage of its unique transmission cycle.