A central feature of Mycobacterium tuberculosis (Mtb) pathogenesis is the ability of Mtb to survive within macrophages (MØ). Despite its critical importance, our appreciation of the interplay between these two cells remains superficial. We employed microarrays to conduct a stepwise dissection of Mtb-MØ interaction during the invasion of resting bone marrow MØ. Contrary to many bacterial pathogens, engagement by MØ receptors without internalization did not alter Mtb gene expression. Subsequently, a high-resolution profile of Mtb invasion-linked gene expression was generated by assaying the Mtb transcriptome at 20 min intervals up to 2 hr postinfection. Transcriptional responses were detected within minutes of phagocytosis, including gene subsets with distinct temporal profiles. Pharmacological manipulation of phagosomal pH and in vitro acid stress studies revealed that vacuole acidification is an important trigger for differential gene expression. Finally, there are marked species-specific differences in the response of Mtb and M. bovis BCG to intraphagosomal cues.