Chlamydia pneumoniae is an important human respiratory pathogen that is responsible for an estimated 10% of community-acquired pneumonia and 5% of bronchitis and sinusitis cases. We examined changes in global protein expression profiles associated with the re-differentiation of RB to EB as C. pneumoniae cells progressed from 24 to 48 hours post-infection in HEp2 cells. Proteins corresponding to those showing the greatest changes in abundance in the beginning of the RB to EB transition were then identified from purified EBs. Among the 300 spots recognized, thirty-five proteins that were expressed at sufficiently high levels were identified by mass spectrometry. We identified C. pneumoniae proteins that showed more than two-fold increases in abundance in the early stages of RB to EB transition, including several associated with amino acid and co-factor biosynthesis (Ndk, TrxA, Adk, PyrH, BirA), maintenance of cytoplasmic protein function (GroEL/ES, DnaK, DksA, GrpE, HtrA, ClpP, ClpB, and Map), modification of the bacterial cell surface (CrpA, OmpA, OmcB), energy metabolism (Tal, Pyk), and the putative transcriptional regulator TctD. This study identified C. pneumoniae proteins involved in the process of re-differentiation into mature, infective EB’s, and indicates bacterial metabolic pathways that may be involved in this transition. The proteins involved in RB to EB transition are key to C. pneumoniae infection and are perhaps suitable targets for therapeutic intervention.