ABSTRACTParachlamydia acanthamoebaeis aChlamydia-related organism whose pathogenic role in pneumonia is supported by serological and molecular clinical studies and an experimental mouse model of lung infection. Toll-like receptors (TLRs) play a seminal role in sensing microbial products and initiating innate immune responses. The aim of this study was to investigate the roles of MyD88, TLR2, and TLR4 in the interaction ofParachlamydiawith macrophages. Here, we showed thatParachlamydiaentered bone-marrow derived macrophages (BMDMs) in a TLR-independent manner but did not multiply intracellularly. Interestingly, compared to live bacteria, heat-inactivatedParachlamydiainduced the production of substantial amounts of tumor necrosis factor alpha (TNF), interleukin-6 (IL-6), and IL-12p40 by BMDMs and of TNF and IL-6 by peritoneal macrophages as well as RAW 264.7 and J774 macrophage cell lines. Cytokine production by BMDMs, which was partially inhibited upon trypsin treatment ofParachlamydia, was dependent on MyD88, TLR4, and, to a lesser extent, TLR2. Finally, MyD88−/−, TLR4−/−, and TLR2−/−mice were as resistant as wild-type mice to lung infection following the intratracheal instillation ofParachlamydia. Thus, in contrast toChlamydia pneumoniae,Parachlamydia acanthamoebaeweakly stimulates macrophages, potentially compensating for its low replication capacity in macrophages by escaping the innate immune surveillance.